Aminoglycoside derivatives and uses thereof in treating genetic disorders

ABSTRACT

Novel aminoglycosides, represented by Formulae Ia and Ib, as defined in the instant specification, designed to exhibit stop codon mutation readthrough activity, are provided. Also provided are pharmaceutical compositions containing the same, and uses thereof in the treatment of genetic diseases and disorders, such as diseases and disorders associated with stop codon mutations.

RELATED APPLICATIONS

This application is a National Phase of PCT Patent Application No.PCT/IL2016/050968 having International filing date of Sep. 2, 2016,which claims the benefit of priority under 35 USC § 119(e) of U.S.Provisional Patent Application Nos. 62/213,143 and 62/213,187 both filedon Sep. 2, 2015 and U.S. Provisional Patent Application No. 62/274,915filed on Jan. 5, 2016. The contents of the above applications are allincorporated by reference as if fully set forth herein in theirentirety.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates toaminoglycosides and more particularly, but not exclusively, to novelaminoglycoside derivatives and their use in increasing an expression ofa gene having a stop codon mutation and/or in the treatment of geneticdisorders.

Many human genetic disorders result from nonsense mutations, where oneof the three stop codons (UAA, UAG or UGA) replaces an amino acid-codingcodon, leading to premature termination of the translation andeventually to truncated inactive proteins. Currently, hundreds of suchnonsense mutations are known, and several were shown to account forcertain cases of fatal diseases, including, for example, cystic fibrosis(CF), Duchenne muscular dystrophy (DMD), ataxia-telangiectasia, Hurlersyndrome, hemophilia A, hemophilia B, Tay-Sachs, Rett Syndrome, UsherSyndrome, Severe epidermolysis bullosa and more. For many of thosediseases there is presently no effective treatment.

Some aminoglycoside compounds have been shown to have therapeutic valuein the treatment of several genetic diseases because of their ability toinduce ribosomes to read-through stop codon mutations, generatingfull-length proteins from part of the mRNA molecules.

Aminoglycosides are highly potent, broad-spectrum antibiotics commonlyused for the treatment of life-threatening infections. It is acceptedthat the mechanism of action of aminoglycoside antibiotics, such asparomomycin (see, FIG. 1), involves interaction with the prokaryoticribosome, and, more specifically, involves binding to the decodingA-site of the 16S ribosomal RNA, which leads to protein translationinhibition and interference with the translational fidelity.

Several achievements in bacterial ribosome structure determination,along with crystal and NMR structures of bacterial A-siteoligonucleotide models, have provided useful information forunderstanding the decoding mechanism in prokaryote cells andunderstanding how aminoglycosides exert their deleterious misreading ofthe genetic code. These studies and others have given rise to thehypothesis that the affinity of the A-site for a non-cognate mRNA-tRNAcomplex is increased upon aminoglycoside binding, preventing theribosome from efficiently discriminating between non-cognate and cognatecomplexes.

The enhancement of termination suppression by aminoglycosides ineukaryotes is thought to occur in a similar mechanism to theaminoglycosides' activity in prokaryotes of interfering withtranslational fidelity during protein synthesis, namely the binding ofcertain aminoglycosides to the ribosomal A-site probably induceconformational changes that stabilize near-cognate mRNA-tRNA complexes,instead of inserting the release factor. Aminoglycosides have been shownto suppress various stop codons with notably different efficiencies(UGA>UAG>UAA), and the suppression effectiveness has been found to befurther dependent upon the identity of the fourth nucleotide immediatelydownstream from the stop codon (C>U>A≥grams) as well as the localsequence context around the stop codon.

The desired characteristics of an effective read-through drug would beoral administration and little or no effect on bacteria. Antimicrobialactivity of read-through drug is undesirable as any unnecessary use ofantibiotics, particularly with respect to the gastrointestinal (GI)biota, due to the adverse effects caused by upsetting the GI biotaequilibrium and the emergence of resistance. In this respect, inaddition to the abovementioned limitations, the majority of clinicalaminoglycosides are greatly selective against bacterial ribosomes, anddo not exert a significant effect on cytoplasmic ribosomes of humancells.

In an effort to circumvent the abovementioned limitations, thebiopharmaceutical industry is seeking new stop codon mutationssuppression drugs by screening large chemical libraries for nonsenseread-through activity.

The first experiments of aminoglycoside-mediated suppression of cysticfibrosis transmembrane conductance regulator protein (CFTR) stop codonmutations demonstrated that premature stop codon mutations found in theCFTR gene could be suppressed by members of the gentamicin family andGeniticin® (G-418) (see, FIG. 1), as measured by the appearance offull-length, functional CFTR in bronchial epithelial cell lines.

Suppression experiments of intestinal tissues from CFTR−/− transgenicmice mutants carrying a human CFTR-G542X transgene showed that treatmentwith gentamicin, and to lesser extent tobramycin, have resulted in theappearance of human CFTR protein at the glands of treated mice. Mostimportantly, clinical studies using double-blind, placebo-controlled,crossover trails have shown that gentamicin can suppress stop codonmutations in affected patients, and that gentamicin treatment improvedtransmembrane conductance across the nasal mucosa in a group of 19patients carrying CFTR stop codon mutations. Other genetic disorders forwhich the therapeutic potential of aminoglycosides was tested inin-vitro systems, cultured cell lines, or animal models include DMD,Hurler syndrome, nephrogenic diabetes insipidus, nephropathiccystinosis, retinitis pigmentosa, and ataxia-telangiectasia.

However, one of the major limitations in using aminoglycosides aspharmaceuticals is their high toxicity towards mammals, typicallyexpressed in kidney (nephrotoxicity) and ear-associated (ototoxicity)illnesses. The origin of this toxicity is assumed to result from acombination of different factors and mechanisms such as interactionswith phospholipids, inhibition of phospholipases and the formation offree radicals. Although considered selective to bacterial ribosomes,most aminoglycosides bind also to the eukaryotic A-site but with loweraffinities than to the bacterial A-site. The inhibition of translationin mammalian cells is also one of the possible causes for the hightoxicity of these agents. Another factor adding to their cytotoxicity istheir binding to the mitochondrial ribosome at the 12S rRNA A-site,whose sequence is very close to the bacterial A-site.

Many studies have been attempted to understand and offer ways toalleviate the toxicity associated with aminoglycosides, including theuse of antioxidants to reduce free radical levels, as well as the use ofpoly-L-aspartate and daptomycin, to reduce the ability ofaminoglycosides to interact with phospholipids. The role of megalin (amultiligand endocytic receptor which is especially abundant in thekidney proximal tubules and the inner ear) in the uptake ofaminoglycosides has recently been demonstrated. The administration ofagonists that compete for aminoglycoside binding to megalin alsoresulted in a reduction in aminoglycoside uptake and toxicity. Inaddition, altering the administration schedule and/or the manner inwhich aminoglycosides are administered has been investigated as means toreduce toxicity.

Despite extensive efforts to reduce aminoglycoside toxicity, few resultshave matured into standard clinical practices and procedures for theadministration of aminoglycosides to suppress stop codon mutations,other than changes in the administration schedule. For example, the useof sub-toxic doses of gentamicin in the clinical trials probably causedthe reduced read-through efficiency obtained in the in-vivo experimentscompared to the in-vitro systems. The aminoglycoside Geneticin® (alsoknown as G-418 sulfate or simply G-418, see, FIG. 1) showed the besttermination suppression activity in in-vitro translation-transcriptionsystems, however, its use as a therapeutic agent is not possible sinceit is lethal even at very low concentrations. For example, the LD₅₀ ofG-418 against human fibroblast cells is 0.04 mg/ml, compared to 2.5-5.0mg/ml for gentamicin, neomycin and kanamycin.

The increased sensitivity of eukaryotic ribosomes to some aminoglycosidedrugs, such as G-418 and gentamicin, is intriguing but up to date couldnot be rationally explained because of the lack of sufficient structuraldata on their interaction with eukaryotic ribosomes. Since G-418 isextremely toxic even at very low concentrations, presently gentamicin isthe only aminoglycoside tested in various animal models and clinicaltrials. Although some studies have shown that due to their relativelylower toxicity in cultured cells, amikacin and paromomycin can representalternatives to gentamicin for stop codon mutation suppression therapy,no clinical trials with these aminoglycosides have been reported yet.

To date, nearly all suppression experiments have been performed withclinical, commercially available aminoglycosides, however, only alimited number of aminoglycosides, including gentamicin, amikacin, andtobramycin, are in clinical use as antibiotics for internaladministration in humans. Among these, tobramycin do not have stop codonmutations suppression activity, and gentamicin is the onlyaminoglycoside tested for stop codon mutations suppression activity inanimal models and clinical trials. Recently, a set of neaminederivatives were shown to promote read-through of the SMN protein infibroblasts derived from spinal muscular atrophy (SPA) patients;however, these compounds were originally designed as antibiotics and noconclusions were derived for further improvement of the read-throughactivity of these derivatives.

WO 2007/113841 and WO 2012/066546 disclose classes ofparomomycin-derived aminoglycosides, designed to exhibit high prematurestop codon mutations readthrough activity while exerting lowcytotoxicity in mammalian cells and low antimicrobial activity, and canthus be used in the treatment of genetic diseases. This class ofparomomycin-derived aminoglycosides was designed by introducing certainmanipulations to the paromamine core, which lead to enhanced readthroughactivity and reduced toxicity and antimicrobial activity. Themanipulations were made on several positions of the paromamine core.

Exemplary such manipulations of the paromamine core which have beentaught in these publications include a hydroxyl group at position 6′ ofthe aminoglycoside core; introduction of one or more monosaccharidemoieties or an oligosaccharide moiety at position 3′, 5 and/or 6 of theaminoglycoside core; introduction of an (S)-4-amino-2-hydroxybutyryl(AHB) moiety at position 1 of the paromamine core; substitution ofhydrogen at position 6′ by an alkyl such as a methyl substituent; and anintroductions of an alkyl group at the 5″ position.

Additional background art includes Nudelman, I., et al., Bioorg Med ChemLett, 2006. 16(24): p. 6310-5; Hobbie, S. N., et al., Nucleic Acids Res,2007. 35(18): p. 6086-93; Kondo, J., et al., Chembiochem, 2007. 8(14):p. 1700-9; Rebibo-Sabbah, A., et al., Hum Genet, 2007. 122(3-4): p.373-81; Azimov, R., et al., Am J Physiol Renal Physiol, 2008. 295(3): p.F633-41; Hainrichson, M., et al., Org Biomol Chem, 2008. 6(2): p.227-39; Hobbie, S. N., et al., Proc Natl Acad Sci USA, 2008. 105(52): p.20888-93; Hobbie, S. N., et al., Proc Natl Acad Sci USA, 2008. 105(9):p. 3244-9; Nudelman, I., et al., Adv. Synth. Catal., 2008. 350: p.1682-1688; Nudelman, I., et al., J Med Chem, 2009. 52(9): p. 2836-45;Venkataraman, N., et al., PLoS Biol, 2009. 7(4): p. e95; Brendel, C., etal., J Mol Med (Berl), 2010. 89(4): p. 389-98; Goldmann, T., et al.,Invest Ophthalmol Vis Sci, 2010. 51(12): p. 6671-80; Malik, V., et al.,Ther Adv Neurol Disord, 2010. 3(6): p. 379-89; Nudelman, I., et al.,Bioorg Med Chem, 2010. 18(11): p. 3735-46; Warchol, M. E., Curr OpinOtolaryngol Head Neck Surg, 2010. 18(5): p. 454-8; Lopez-Novoa, J. M.,et al., Kidney Int, 2011. 79(1): p. 33-45; Rowe, S. M., et al., J MolMed (Berl), 2011. 89(11): p. 1149-61; Vecsler, M., et al., PLoS One,2011. 6(6): p. e20733; U.S. Pat. Nos. 3,897,412, 4,024,332, 4,029,882,and 3,996,205; Greenberg et al., J. Am. Chem. Soc., 1999, 121,6527-6541; Kotra et al., antimicrobial agents and chemotherapy, 2000, p.3249-3256; Haddad et al., J. Am. Chem. Soc., 2002, 124, 3229-3237;Kandasamy, J. et al., J. Med. Chem. 2012, 55, pp. 10630-10643; Duscha,S. et al., M Bio, 2014, 5(5), p. e01827-14; Huth, M. E. et al., J ClinInvest., 2015, 125(2), pp. 583-92; Shulman, E. et al., J Biol Chem.,2014, 289(4), pp. 2318-30 and FR Patent No. 2,427,341, JP Patent No.04046189. The teachings of all of these documents are incorporated byreference as if fully set forth herein.

SUMMARY OF THE INVENTION

The present invention relates to aminoglycosides, which can bebeneficially used in the treatment of genetic diseases, by exhibitinghigh premature stop codon mutations read-through activity, low toxicityin mammalian cells and low antimicrobial activity, as well as improvedbioavailability and/or cell permeability. The presently disclosedaminoglycosides are characterized by a core structure based on Rings I,II and optionally III of paromomycin.

According to an aspect of some embodiments of the present inventionthere is provided a compound represented by Formula I:

or a pharmaceutically acceptable salt thereof,

wherein:

the dashed line indicates a stereo-configuration of position 6′ being anR configuration or an S configuration;

X₁ is O or S;

the dashed bond between C4′ and C5′ in Ring I represents a single bondor a double bond;

the dashed bond between C4′ and C3′ in Ring I represents a single bondor a double bond;

Rx, Ry1 and Rz are each independently hydrogen, alkyl or cycloalkyl, orabsent, wherein at least Rz is absent in case the dashed bond betweenC4′ and C5′ is a double bond, and wherein at least Ry1 is absent in casethe dashed bond between C4′ and C3′ is a double bond;

Ry2-Ry9 and Rw1-Rw3 are each independently selected from hydrogen,alkyl, alkenyl, alkynyl, aryl, heteroaryl and cycloalkyl, each beingsubstituted or unsubstituted, or, alternatively, each can be as definedherein for R₇-R₉;

R₁ is selected from the group consisting of hydrogen, a substituted orunsubstituted alkyl, a substituted or unsubstituted alkenyl, asubstituted or unsubstituted alkynyl, a substituted or unsubstitutedcycloalkyl, a substituted or unsubstituted aryl, a substituted orunsubstituted heteroaryl, a substituted or unsubstituted alkaryl, asubstituted or unsubstituted amine, a substituted or unsubstitutedamide, an acyl, a carboxylate, and a saturated or unsaturated and/orsubstituted or unsubstituted hydroxy alkyl (e.g., —CH₂—OH);

R₂ is selected from the group consisting of hydrogen, a substituted orunsubstituted alkyl, a substituted or unsubstituted alkenyl, asubstituted or unsubstituted alkynyl, a substituted or unsubstitutedcycloalkyl, a substituted or unsubstituted aryl, a substituted orunsubstituted heteroaryl, a substituted or unsubstituted alkaryl andacyl;

R₃ and R₄ are each independently selected from the group consisting ofhydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heteroalicyclic, aryl,heteroaryl, amine and OR₁₆, wherein R₁₆ is independently selected fromhydrogen, a monosaccharide moiety, an oligosaccharide moiety, asubstituted or unsubstituted alkyl, a substituted or unsubstitutedalkenyl, a substituted or unsubstituted alkynyl, a substituted orunsubstituted cycloalkyl, a substituted or unsubstituted aryl, asubstituted or unsubstituted heteroaryl, a substituted or unsubstitutedalkaryl and acyl, or is absent, wherein R₃ is optionally absent in casethe dashed bond between C4′ and C5′ is a double bond, and R₄ isoptionally absent in case the dashed bond between C4′ and C3′ is adouble bond;

R₅ and R₆ are each independently selected from the group consisting ofhydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heteroalicyclic, aryl,heteroaryl, amine and OR₁₆, wherein R₁₆ is independently selected fromhydrogen, a monosaccharide moiety, an oligosaccharide moiety, asubstituted or unsubstituted alkyl, a substituted or unsubstitutedalkenyl, a substituted or unsubstituted alkynyl, a substituted orunsubstituted cycloalkyl, a substituted or unsubstituted aryl, asubstituted or unsubstituted heteroaryl, a substituted or unsubstitutedalkaryl and acyl; and

R₇-R₉ are each independently selected from the group consisting ofhydrogen, acyl, an amino-substituted alpha-hydroxy acyl, a substitutedor unsubstituted alkyl, a substituted or unsubstituted alkenyl, asubstituted or unsubstituted alkynyl, a substituted or unsubstitutedcycloalkyl, a substituted or unsubstituted aryl, a substituted orunsubstituted alkaryl, carboxylate, sulfonyl (including alkyl sulfonyland aryl sulfonyl) and a cell-permealizable group as defined herein.

It is to be noted that herein throughout, the stereoconfiguration ofRings I, II and III, if present, can be any possible, compatibleconfiguration, and are therefore not to be limited to the illustrationof these rings in the general Formulae presented herein. Exemplarystereroconfigurations are presented hereinunder.

According to some of any of the embodiments described herein, at leastone of R₃, R₄, R₅ and R₆ is OR₁₆.

According to some of any of the embodiments described herein, R₁₆ is anaryl.

According to some of any of the embodiments described herein, at leastone of R₃, R₄, R₅ and R₆ is selected from the group consisting ofphenyloxy, 1-anthryloxy, 1-naphthyloxy, 2-naphthyloxy, 2-phenanthryloxyand 9-phenanthryloxy.

According to some of any of the embodiments described herein, R₁₆ is asubstituted or unsubstituted heteroaryl, and at least one of R₃, R₄, R₅and R₆ is independently a substituted or unsubstituted heteroaryloxy.

According to some of any of the embodiments described herein, at leastone of R₃, R₄, R₅ and R₆ is independently selected from the groupconsisting of 2-anthryloxy, 2-furyloxy, 2-indolyloxy, 2-naphthyloxy,2-pyridyloxy, 2-pyrimidyloxy, 2-pyrryloxy, 2-quinolyloxy, 2-thienyloxy,3-furyloxy, 3-indolyloxy, 3-thienyloxy, 4-imidazolyloxy, 4-pyridyloxy,4-pyrimidyloxy, 4-quinolyloxy, 5-methyl-2-thienyloxy and6-chloro-3-pyridyloxy.

According to some of any of the embodiments described herein, R₁₆ is asubstituted aryl.

According to some of any of the embodiments described herein, at leastone of R₃, R₄, R₅ and R₆ is OR₁₆, and R₁₆ is independently selected fromthe group consisting of 2-(N-ethylamino)phenyl, 2-(N-hexylamino)phenyl,2-(N-methylamino)phenyl, 2,4-dimethoxyphenyl, 2-acetamidophenyl,2-aminophenyl, 2-carboxyphenyl, 2-chlorophenyl, 2-ethoxyphenyl,2-fluorophenyl, 2-hydroxymethylphenyl, 2-hydroxyphenyl, 2-hydroxyphenyl,2-methoxycarbonylphenyl, 2-methoxyphenyl, 2-methylphenyl,2-N,N-dimethylaminophenyl, 2-trifluoromethylphenyl,3-(N,N-dibutylamino)phenyl, 3-(N,N-diethylamino)phenyl,3,4,5-trimethoxyphenyl, 3,4-dichlorophenyl, 3,4-dimethoxyphenyl,3,5-dimethoxyphenyl, 3-aminophenyl, 3-biphenylyl, 3-carboxyphenyl,3-chloro-4-methoxyphenyl, 3-chlorophenyl, 3-ethoxycarbonylphenyl,3-ethoxyphenyl, 3-fluorophenyl, 3-hydroxymethylphenyl, 3-hydroxyphenyl,3-isoamyloxyphenyI, 3-isobutoxyphenyl, 3-isopropoxyphenyl,3-methoxyphenyl, 3-methylphenyl, 3-N,N-dimethylaminophenyl, 3-tolyl,3-trifluoromethylphenyl, 4-(benzyloxy)phenyl,4-(isopropoxycarbonyl)phenyl, 4-(N,N-diethylamino)phenyl,4-(N,N-dihexylamino)phenyl, 4-(N,N-diisopropylamino)phenyl,4-(N,N-dimethylamino)phenyl, 4-(N,N-di-n-pentylamino)phenyl,4-(n-hexyloxycarbonyl)phenyl, 4-(N-methylamino)phenyl,4-(trifluoromethyl)phenyl, 4-aminophenyl, 4-benzyloxyphenyl,4-biphenylyl, 4-butoxyphenyl, 4-butyramidophenyl, 4-carboxyphenyl,4-chlorophenyl, 4-ethoxycarbonylphenyl, 4-hexanamidophenyl,4-hydroxymethylphenyl, 4-hydroxyphenyl, 4-iodophenyl, 4-isobutylphenyl,4-isobutyramidophenyl, 4-isopropoxyphenyl, 4-isopropylphenyl,4-methoxyphenyl, 4-methylphenyl, 4-n-hexanamidophenyl,4-n-hexyloxyphenyl, 4-n-hexylphenyl, 4-nitrophenyl, 4-nitrophenyl,4-propionamidophenyl, 4-tolyl, 4-trifluoromethylphenyl and4-valeroyloxycarbonylphenyl.

According to some of any of the embodiments described herein, R₃ is OR₁₆and R₁₆ is hydrogen.

According to some of any of the embodiments described herein, R₃ is OR₁₆and R₁₆ is selected from the group consisting of methyl, ethyl, propyl,butyl, pentyl, propenyl, 2-hydroxyethyl, 3-hydroxypropyl,2,3-dihydroxypropyl and methoxymethyl.

According to some of any of the embodiments described herein, at leastone, or each, of R₃, R₄, R₅ and R₆ is OR₁₆ and R₁₆ is independently anacyl.

According to some of any of the embodiments described herein, each ofR₃, R₄, R₅ and R₆ is OR₁₆ and R₁₆ is hydrogen.

According to some of any of the embodiments described herein, at leastone of R₃, R₄, R₅ and —R₆ is OR₁₆ in which R₁₆ is the monosaccharidemoiety.

According to some of any of the embodiments described herein, themonosaccharide moiety is represented by Formula II:

wherein:

the curved line denotes a position of attachment;

the dashed line indicates a stereo-configuration of position 5″ being anR configuration or an S configuration;

X₂ is OR₁₃ or NR₁₄R₁₅;

each of R₁₀, R₁₁ and R₁₃ is independently selected from the groupconsisting of hydrogen, a substituted or unsubstituted alkyl, asubstituted or unsubstituted alkenyl, a substituted or unsubstitutedalkynyl, a substituted or unsubstituted cycloalkyl, a substituted orunsubstituted aryl, a substituted or unsubstituted heteroaryl, asubstituted or unsubstituted alkaryl, and acyl;

R₁₂ is selected from the group consisting of hydrogen, a substituted orunsubstituted alkyl, a substituted or unsubstituted alkenyl, asubstituted or unsubstituted alkynyl, a substituted or unsubstitutedcycloalkyl, a substituted or unsubstituted aryl, a substituted orunsubstituted heteroaryl, a substituted or unsubstituted alkaryl, asubstituted or unsubstituted amine, a substituted or unsubstitutedamide, an acyl, a carboxylate, and a saturated or unsaturated and/orsubstituted or unsubstituted hydroxyalkyl;

each of R₁₄- and R₁₅ is independently selected from the group consistingof hydrogen, a substituted or unsubstituted alkyl, a substituted orunsubstituted alkenyl, a substituted or unsubstituted alkynyl, asubstituted or unsubstituted cycloalkyl, a substituted or unsubstitutedaryl, a substituted or unsubstituted heteroaryl, a substituted orunsubstituted alkaryl, acyl, and a cell-permealizable group, or,alternatively, R₁₄ and R₁₅, when present, form together a heterocyclicring.

Substituents not shown in Formula II at positions such as 6′, 1″, 2″,3″, 4″ and 5″ are typically hydrogen, although other substituents, suchas, but not limited, as defined for Ry2-Ry9, are also contemplated.

According to some of any of the embodiments described herein, thecompound is represented by Formula Ib:

Substituents not shown in Formula Ib at positions such as 6′, 1″, 2″,3″, 4″ and 5″ are typically hydrogen, although other substituents, suchas, but not limited, as defined for Ry2-Ry9, are also contemplated.

According to some of any of the embodiments described herein, X₂ isOR₁₃.

According to some of any of the embodiments described herein, X₂ isNR₁₄R₁₅.

According to some of any of the embodiments described herein, R₁₂ isother than hydrogen.

According to some of any of the embodiments described herein, at leastone of R₁₀, R₁₁ and R₁₃ if present is an acyl.

According to some of any of the embodiments described herein, X₁ is O.

According to some of any of the embodiments described herein, the bondbetween C4′ and C5′ in Ring I is a single bond.

According to some of any of the embodiments described herein, the bondbetween C4′ and C5′ in Ring I is a double bond and Rx or R₃, and Rz, areabsent.

According to some of any of the embodiments described herein, the bondbetween C4′ and C3′ in Ring I is a single bond.

According to some of any of the embodiments described herein, the bondbetween C4′ and C3′ in Ring I is a double bond and Rx or R₄, and Ry1,are absent.

According to some of any of the embodiments described herein, R₁ isother than hydrogen.

According to some of any of the embodiments described herein, R₁ is ahydroxyalkyl.

According to some of any of the embodiments described herein, R₁ is ahydroxymethyl.

According to some of any of the embodiments described herein, R₁ is orcomprises a substituted or unsubstituted alkyl, a substituted orunsubstituted alkenyl or a substituted or unsubstituted alkynyl.

According to some of any of the embodiments described herein, R₁ isselected from the group consisting of methyl, ethyl, propyl, butyl andpentyl.

According to some of any of the embodiments described herein, R₁ is orcomprises an aryl.

According to some of any of the embodiments described herein, R₁ isselected from the group consisting of phenyl, 1-anthryl, 1-naphthyl,2-naphthyl, 2-phenanthryl and 9-phenanthryl.

According to some of any of the embodiments described herein, R₁ is orcomprises a substituted or unsubstituted heteroaryl.

According to some of any of the embodiments described herein, R₁ isselected from the group consisting of 2-anthryl, 2-furyl, 2-indolyl,2-naphthyl, 2-pyridyl, 2-pyrimidyl, 2-pyrryl, 2-quinolyl, 2-thienyl,3-furyl, 3-indolyl, 3-thienyl, 4-imidazolyl, 4-pyridyl, 4-pyrimidyl,4-quinolyl, 5-methyl-2-thienyl and 6-chloro-3-pyridyl.

According to some of any of the embodiments described herein, R₁ is orcomprises a substituted aryl.

According to some of any of the embodiments described herein, R₁ isselected from the group consisting of 2-(N-ethylamino)phenyl,2-(N-hexylamino)phenyl, 2-(N-methylamino)phenyl, 2,4-dimethoxyphenyl,2-acetamidophenyl, 2-aminophenyl, 2-carboxyphenyl, 2-chlorophenyl,2-ethoxyphenyl, 2-fluorophenyl, 2-hydroxymethylphenyl, 2-hydroxyphenyl,2-hydroxyphenyl, 2-methoxycarbonylphenyl, 2-methoxyphenyl,2-methylphenyl, 2-N,N-dimethylaminophenyl, 2-trifluoromethylphenyl,3-(N,N-dibutylamino)phenyl, 3-(N,N-diethylamino)phenyl,3,4,5-trimethoxyphenyl, 3,4-dichlorophenyl, 3,4-dimethoxyphenyl,3,5-dimethoxyphenyl, 3-aminophenyl, 3-biphenylyl, 3-carboxyphenyl,3-chloro-4-methoxyphenyl, 3-chlorophenyl, 3-ethoxycarbonylphenyl,3-ethoxyphenyl, 3-fluorophenyl, 3-hydroxymethylphenyl, 3-hydroxyphenyl,3-isoamyloxyphenyl, 3-isobutoxyphenyl, 3-isopropoxyphenyl,3-methoxyphenyl, 3-methylphenyl, 3-N,N-dimethylaminophenyl, 3-tolyl,3-trifluoromethylphenyl, 4-(benzyloxy)phenyl,4-(isopropoxycarbonyl)phenyl, 4-(N,N-diethylamino)phenyl,4-(N,N-dihexylamino)phenyl, 4-(N,N-diisopropylamino)phenyl,4-(N,N-dimethylamino)phenyl, 4-(N,N-di-n-pentylamino)phenyl,4-(n-hexyloxycarbonyl)phenyl, 4-(N-methylamino)phenyl,4-(trifluoromethyl)phenyl, 4-aminophenyl, 4-benzyloxyphenyl,4-biphenylyl, 4-butoxyphenyl, 4-butyramidophenyl, 4-carboxyphenyl,4-chlorophenyl, 4-ethoxycarbonylphenyl, 4-hexanamidophenyl,4-hydroxymethylphenyl, 4-hydroxyphenyl, 4-iodophenyl, 4-isobutylphenyl,4-isobutyramidophenyl, 4-isopropoxyphenyl, 4-isopropylphenyl,4-methoxyphenyl, 4-methylphenyl, 4-n-hexanamidophenyl,4-n-hexyloxyphenyl, 4-n-hexylphenyl, 4-nitrophenyl, 4-nitrophenyl,4-propionamidophenyl, 4-tolyl, 4-trifluoromethylphenyl and4-valeroyloxycarbonylphenyl.

According to some of any of the embodiments described herein, R₁ is orcomprises an amine.

According to some of any of the embodiments described herein, R₁ isselected from the group consisting of —NH₂, —NHCH₃, —N(CH₃)₂,—NH—CH₂—CH₂—NH₂, —NH—CH₂—CH₂—OH and —NH—CH₂—CH(OCH₃)₂.

According to some of any of the embodiments described herein, R₂ ishydrogen.

According to some of any of the embodiments described herein, R₂ isalkyl, preferably selected from the group consisting of methyl, ethyland propyl.

According to some of any of the embodiments described herein, R₂ isacyl.

According to some of any of the embodiments described herein, R₇ isselected from the group consisting of hydrogen,(R/S)-4-amino-2-hydroxybutyryl (AHB), (R/S)-3-amino-2-hydroxypropionate(AHP), (R/S)-3-amino-2-hydroxypropionyl, 5-aminopentanoyl,5-hydroxypentanoyl, formyl, —C(═O)—O-methyl, —C(═O)—O-ethyl,—C(═O)—O-benzyl, -β-amino-α-hydroxypropionyl, -δ-amino-α-hydroxyvaleryl,-β-benzyloxycarbonylamino-α-hydroxypropionyl,-δ-benzyloxycarbonylamino-α-hydroxyvaleryl, methylsulfonyl,phenylsulfonyl, benzoyl, propyl, isopropyl, —(CH₂)₂NH₂, —(CH₂)₃NH₂,—CH₂CH(NH₂)CH₃, —(CH₂)₄NH₂, —(CH₂)₅NH₂, —(CH₂)₂NH-ethyl,—(CH₂)₂NH(CH₂)₂NH₂, —(CH₂)₃NH(CH₂)₃NH₂, —(CH₂)₃NH(CH₂)₄NH(CH₂)₃NH₂,—CH(—NH₂)CH₂(OH), —CH(—OH)CH₂(NH₂), —CH(—OH)—(CH₂)₂(NH₂),—CH(—NH₂)—(CH₂)₂(OH), —CH(—CH₂NH₂)—(CH₂OH), —(CH₂)₄NH(CH₂)₃NH₂,—(CH₂)₂NH(CH₂)₂NH(CH₂)₂NH₂, —(CH₂)₂N(CH₂CH₂NH₂)₂, —CH₂—C(═O)NH₂,—CH(CH₃)—C(═O)NH₂, —CH₂-phenyl, —CH(i-propyl)-C(═O)NH₂,—CH(benzyl)-C(═O)NH₂, —(CH₂)₂₀H, —(CH₂)₃OH and —CH(CH₂OH)₂.

According to some of any of the embodiments described herein, each of R₈and R₉ is independently selected from the group consisting of hydrogen,(R/S)-4-amino-2-hydroxybutyryl (AHB), (R/S)-3-amino-2-hydroxypropionate(AHP), (R/S)-3-amino-2-hydroxypropionyl, 5-aminopentanoyl,5-hydroxypentanoyl, formyl, —COO-methyl, —COO-ethyl, —COO-benzyl,-β-β-amino-α-hydroxypropionyl, -δ-amino-α-hydroxyvaleryl,-β-benzyloxycarbonylamino-α-hydroxypropionyl,-δ-benzyloxycarbonylamino-α-hydroxyvaleryl, methylsulfonyl,phenylsulfonyl, benzoyl, propyl, isopropyl, —(CH₂)₂NH₂, —(CH₂)₃NH₂,—CH₂CH(NH₂)CH₃, —(CH₂)₄NH₂, —(CH₂)₅NH₂, —(CH₂)₂NH-ethyl,—(CH₂)₂NH(CH₂)₂NH₂, —(CH₂)₃NH(CH₂)₃NH₂, —(CH₂)₃NH(CH₂)₄NH(CH₂)₃NH₂,—CH(—NH₂)CH₂(OH), —CH(—OH)CH₂(NH₂), —CH(—OH)—(CH₂)₂(NH₂),—CH(—NH₂)—(CH₂)₂(OH), —CH(—CH₂NH₂)—(CH₂OH), —(CH₂)₄NH(CH₂)₃NH₂,—(CH₂)₂NH(CH₂)₂NH(CH₂)₂NH₂, —(CH₂)₂N(CH₂CH₂NH₂)₂, —CH₂—C(═O)NH₂,—CH(CH₃)—C(═O)NH₂, —CH₂-phenyl, —CH(i-propyl)-C(═O)NH₂,—CH(benzyl)-C(═O)NH₂, —(CH₂)₂₀H, —(CH₂)₃OH and —CH(CH₂OH)₂.

According to some of any of the embodiments described herein, theamino-substituted alpha-hydroxy acyl is (S)-4-amino-2-hydroxybutyryl(AHB).

According to some of any of the embodiments described herein, thecell-permealizable group is guanidyl.

According to some of any of the embodiments described herein, anunsubstituted aryl as described herein in any of the respectiveembodiments is selected from the group consisting of phenyl, 1-anthryl,1-naphthyl, 2-naphthyl, 2-phenanthryl and 9-phenanthryl.

According to some of any of the embodiments described herein, asubstituted or unsubstituted heteroaryl as described herein in any ofthe respective embodiments is selected from the group consisting of2-anthryl, 2-furyl, 2-indolyl, 2-naphthyl, 2-pyridyl, 2-pyrimidyl,2-pyrryl, 2-quinolyl, 2-thienyl, 3-furyl, 3-indolyl, 3-thienyl,4-imidazolyl, 4-pyridyl, 4-pyrimidyl, 4-quinolyl, 5-methyl-2-thienyl and6-chloro-3-pyridyl.

According to some of any of the embodiments described herein, asubstituted aryl as described herein in any of the respectiveembodiments is selected from the group consisting of2-(N-ethylamino)phenyl, 2-(N-hexylamino)phenyl, 2-(N-methylamino)phenyl,2,4-dimethoxyphenyl, 2-acetamidophenyl, 2-aminophenyl, 2-carboxyphenyl,2-chlorophenyl, 2-ethoxyphenyl, 2-fluorophenyl, 2-hydroxymethylphenyl,2-hydroxyphenyl, 2-hydroxyphenyl, 2-methoxycarbonylphenyl,2-methoxyphenyl, 2-methylphenyl, 2-N,N-dimethylaminophenyl,2-trifluoromethylphenyl, 3-(N,N-dibutylamino)phenyl,3-(N,N-diethylamino)phenyl, 3,4,5-trimethoxyphenyl, 3,4-dichlorophenyl,3,4-dimethoxyphenyl, 3,5-dimethoxyphenyl, 3-aminophenyl, 3-biphenylyl,3-carboxyphenyl, 3-chloro-4-methoxyphenyl, 3-chlorophenyl,3-ethoxycarbonylphenyl, 3-ethoxyphenyl, 3-fluorophenyl,3-hydroxymethylphenyl, 3-hydroxyphenyl, 3-isoamyloxyphenyl,3-isobutoxyphenyl, 3-isopropoxyphenyl, 3-methoxyphenyl, 3-methylphenyl,3-N,N-dimethylaminophenyl, 3-tolyl, 3-trifluoromethylphenyl,4-(benzyloxy)phenyl, 4-(isopropoxycarbonyl)phenyl,4-(N,N-diethylamino)phenyl, 4-(N,N-dihexylamino)phenyl,4-(N,N-diisopropylamino)phenyl, 4-(N,N-dimethylamino)phenyl,4-(N,N-di-n-pentylamino)phenyl, 4-(n-hexyloxycarbonyl)phenyl,4-(N-methylamino)phenyl, 4-(trifluoromethyl)phenyl, 4-aminophenyl,4-benzyloxyphenyl, 4-biphenylyl, 4-butoxyphenyl, 4-butyramidophenyl,4-carboxyphenyl, 4-chlorophenyl, 4-ethoxycarbonylphenyl,4-hexanamidophenyl, 4-hydroxymethylphenyl, 4-hydroxyphenyl,4-iodophenyl, 4-isobutylphenyl, 4-isobutyramidophenyl,4-isopropoxyphenyl, 4-isopropylphenyl, 4-methoxyphenyl, 4-methylphenyl,4-n-hexanamidophenyl, 4-n-hexyloxyphenyl, 4-n-hexylphenyl,4-nitrophenyl, 4-nitrophenyl, 4-propionamidophenyl, 4-tolyl,4-trifluoromethylphenyl and 4-valeroyloxycarbonylphenyl.

According to some of any of the embodiments described herein, an acyl asdescribed herein in any of the respective embodiments is selected fromthe group consisting of a hydrocarbon acyl radical having from 2 to 18carbon atoms, optionally substituted by one or more of halo, nitro,hydroxy, amine, cyano, thiocyano, and alkoxy.

According to some of any of the embodiments described herein, the acylis derived from an acid selected from the group consisting of asaturated or unsaturated and/or substituted or unsubstituted aliphaticcarboxylic acid, acetic acid, propionic acid, butyric acid, isobutyricacid, tert-butylacetic acid, valeric acid, isovaleric acid, caproicacid, caprylic acid, decanoic acid, dodecanoic acid, lauric acid,tridecanoic acid, myristic acid, pentadecanoic acid, palmitic acid,margaric acid, stearic acid, acrylic acid, crotonic acid, undecylenicacid, oleic acid, hexynoic acid, heptynoic acid, octynoic acid, asaturated or unsaturated alicyclic carboxylic acid,cyclobutanecarboxylic acid, cyclopentanecarboxylic acid,cyclopentenecarboxylic acid, methylcyclopentenecarboxylic acid,cyclohexanecarboxylic acid, dimethylcyclohexanecarboxylic acid,dipropylcyclohexanecarboxylic acid, a saturated or unsaturated,alicyclic aliphatic carboxylic acid, cyclopentaneacetic acid,cyclopentanepropionic acid, cyclohexaneacetic acid, cyclohexanebutyricacid, methylcyclohexaneacetic acid, a substituted or unsubstitutedaromatic carboxylic acid, benzoic acid, toluic acid, naphthoic acid,ethylbenzoic acid, isobutylbenzoic acid, methylbutylbenzoic acid, anaromatic aliphatic carboxylic acid, phenylacetic acid, phenylpropionicacid, phenylvaleric acid, cinnamic acid, phenylpropiolic acid,naphthylacetic acid, a halo-alkoxyhydrocarbon carboxylic acid, anitro-alkoxyhydrocarbon carboxylic acid, a hydroxy-alkoxyhydrocarboncarboxylic acid, an amino-alkoxyhydrocarbon carboxylic acid, acyano-alkoxyhydrocarbon carboxylic acid, a thiocyano-alkoxyhydrocarboncarboxylic acid, mono-acetic acid, di-acetic acid, trichloroacetic acid,1,2,3,4,5,6-hexachlorocyclohexanecarboxylic acid,1,2-dibromo-4-methylcyclohexanecarboxylic acid,1,6-dibromo-3-methylcyclohexanecarboxylic acid,1-bromo-3,5-dimethylcyclohexanecarboxylic acid,2-chlorocyclohexanecarboxylic acid, 4-chlorocyclohexanecarboxylic acid,2,3-dibromo-2-methylcyclohexanecarboxylic acid, 2,4,6-trinitrobenzoicacid, 2,5-dibromo-2-methylcyclohexanecarboxylic acid,2-bromo-4-methylcyclohexanecarboxylic acid,2-nitro-1-methyl-cyclobutanecarboxylic acid, 3,4-dinitrobenzoic acid,3,5-dinitrobenzoic acid, 3-bromo-2,2,3-trimethylcyclopentanecarboxylicacid, 3-bromo-2-methylcyclohexanecarboxylic acid,3-bromo-3-methylcyclohexanecarboxylic acid,4-bromo-2-methylcyclohexanecarboxylic acid,5-bromo-2-methylcyclohexanecarboxylic acid, 4,4-dichlorobenzilic acid,4,5-dibromo-2-methylcyclohexanecarboxylic acid,5-bromo-2-methylcyclohexanecarboxylic acid,6-bromo-2-methylcyclohexanecarboxylic acid,5,6-dibromo-2-methylcyclohexanecarboxylic acid,6-bromo-3-methylcyclohexanecarboxylic acid, anisic acid, cyanoaceticacid, cyanopropionic acid, ethoxyformic acid (ethyl hydrogen carbonate),gallic acid, homogentisic acid, o-, m-, and p-chlorobenzoic acid, lacticacid, mevalonic acid, o-, m-, p-nitrobenzoic acid, p-hydroxybenzoicacid, salicylic acid, shikimic acid, thiocyanoacetic acid,trimethoxybenzoic acid, trimethoxycinnamic acid, veratric acid, α- andβ-chloropropionic acid, α- and γ-bromobutyric acid and α- andδ-iodovaleric acid, β-resorcylic acid.

According to an aspect of some embodiments of the present inventionthere is provided a pharmaceutical composition comprising the compoundas described herein in any one of the embodiments and any combinationthereof, and a pharmaceutically acceptable carrier.

According to some of any of the embodiments described herein, thepharmaceutical composition is packaged in a packaging material andidentified in print, in or on the packaging material, for use in thetreatment of a genetic disorder with a premature stop-codon truncationmutation and/or a protein truncation phenotype.

According to an aspect of some embodiments of the present inventionthere is provided a method for treating a genetic disorder with apremature stop-codon truncation mutation and/or a protein truncationphenotype, the method comprising administering to a subject in needthereof a therapeutically effective amount of the compound as describedherein in any one of the embodiments and any combination thereof.

According to an aspect of some embodiments of the present inventionthere is provided a compound as described herein in any one of theembodiments and any combination thereof, for use in the treatment of agenetic disorder with a premature stop-codon truncation mutation and/ora protein truncation phenotype.

According to an aspect of some embodiments of the present inventionthere is provided a use of the compound as described herein in any oneof the embodiments and any combination thereof, in the manufacture of amedicament for treating a genetic disorder with a premature stop-codontruncation mutation and/or a protein truncation phenotype.

According to some of any of the embodiments described herein, thegenetic disorder is selected from the group consisting of cysticfibrosis (CF), Duchenne muscular dystrophy (DMD), ataxia-telangiectasia,Hurler syndrome, hemophilia A, hemophilia B, Usher syndrome, Tay-Sachs,Becker muscular dystrophy (BMD), Congenital muscular dystrophy (CMD),Factor VII deficiency, Familial atrial fibrillation, Hailey-Haileydisease, McArdle disease, Mucopolysaccharidosis, Nephropathiccystinosis, Polycystic kidney disease, Rett syndrome, Spinal muscularatrophy (SMA), cystinosis, Severe epidermolysis bullosa, Dravetsyndrome, X-linked nephrogenic diabetes insipidus (XNDI), X-linkedretinitis pigmentosa and cancer.

According to an aspect of some embodiments of the present inventionthere is provided a method of increasing the expression level of a genehaving a premature stop-codon mutation, the method comprisingtranslating the gene into a protein in the presence of a compound asdescribed herein in any of the respective embodiments and anycombination thereof.

According to an aspect of some embodiments of the present inventionthere is provided a compound as described herein in any of therespective embodiments and any combination thereof for use in increasingthe expression level of a gene having a premature stop-codon mutation.

According to an aspect of some embodiments of the present inventionthere is provided a use of a compound as described herein in any of therespective embodiments and any combination thereof in the manufacture ofa medicament for increasing the expression level of a gene having apremature stop-codon mutation.

According to some of any of the embodiments described herein, thepremature stop-codon mutation has an RNA code selected from the groupconsisting of UGA, UAG and UAA.

According to some of any of the embodiments described herein, theprotein is translated in a cytoplasmic translation system.

According to some of any of the embodiments described herein, thecompound is used in a mutation suppression amount.

According to some of any of the embodiments described herein, aninhibition of translation IC₅₀ of the compound in a eukaryoticcytoplasmic translation system is greater that an inhibition oftranslation IC₅₀ of the compound in a ribosomal translation system.

According to some of any of the embodiments described herein, aninhibition of translation IC₅₀ of the compound in a eukaryoticcytoplasmic translation system is greater that an inhibition oftranslation IC₅₀ of the compound in a prokaryotic translation system.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the invention. In this regard, thedescription taken with the drawings makes apparent to those skilled inthe art how embodiments of the invention may be practiced.

In the drawings:

FIG. 1 (Background Art) presents the chemical structures of some knownfamilies of aminoglycosides;

FIGS. 2A-C present comparative bar plot showing readthrough levels ofthe Rett syndrome causing premature stop codon mutations R168X (FIG.2A), R270X (FIG. 2B) and R294X (FIG. 2C), as measured and calculated forexemplary compounds according to some embodiments of the presentinvention, being contacted with expression HEK293 cells at aconcentration of 0.3 mM and 1 mM, as well as for a control sample (noadded compound), based on the firefly/renilla expression ratios versusthe expression ratios observed in the wild type (WT);

FIGS. 3A-C present comparative bar plot showing readthrough levels ofthe Rett syndrome causing premature stop codon mutations R168X (FIG.3A), R270X (FIG. 3B) and R294X (FIG. 3C), as measured and calculated forexemplary compounds according to some embodiments of the presentinvention, being contacted with expression HEK293 cells at aconcentration of 0.3 mM and 1 mM, as well as for a control sample (noadded compound), and presented as fractions of the firefly/renillaexpression ratios observed for the control sample (100%) and compared tothe expression ratios observed in the WT;

FIGS. 4A-F present the results of cystic fibrosis G542X nonsensemutation suppression dose-response cell-free assays conducted forexemplary compounds according to some embodiments of the presentinvention, NB144, NB145 and NB146, at a concentration range of 0-50 μM,wherein FIG. 4A shows the expression level of the firefly luciferasewhich is found downstream of the WT sequence, FIG. 4B shows theexpression level of the firefly luciferase which is found downstream ofthe G542X mutant sequence, FIG. 4C shows the expression level of therenilla luciferase which is found upstream of the WT sequence, FIG. 4Dshows the expression level of the renilla luciferase which is foundupstream of the G542X mutant sequence, FIG. 4E shows the firefly/renillaexpression ratio measured in the WT sequence, and FIG. 4F shows thefirefly/renilla expression ratio measured in the G542X mutant sequence;

FIGS. 5A-B present the results of cystic fibrosis G542X nonsensemutation suppression dose-response cell-free assays conducted forexemplary compounds according to some embodiments of the presentinvention, NB144, NB145 and NB146, at a concentration rage of 0-50 aM,wherein FIG. 5A shows the expression level of the firefly luciferase,which is found downstream of the mutant sequence, as a fraction of theexpression level exhibited in the control experiment (no addedcompound), and FIG. 5B shows the firefly/renilla expression ratio, downand upstream of the mutant sequence, as a fraction of the expressionlevel in the control experiment;

FIGS. 6A-F present the results of cystic fibrosis G542X nonsensemutation suppression dose-response cell-free assays conducted forexemplary compounds according to some embodiments of the presentinvention, NB150, NB151 and NB152, at a concentration rage of 0-50 aM,wherein FIG. 6A shows the expression level of the firefly luciferasewhich is found downstream of the WT sequence, FIG. 6B shows theexpression level of the firefly luciferase which is found downstream ofthe G542X mutant sequence, FIG. 6C shows the expression level of therenilla luciferase which is found upstream of the WT sequence, FIG. 6Dshows the expression level of the renilla luciferase which is foundupstream of the G542X mutant sequence, FIG. 6E shows the firefly/renillaexpression ratio measured in the WT sequence, and FIG. 6F shows thefirefly/renilla expression ratio measured in the G542X mutant sequence;

FIGS. 7A-B present the results of cystic fibrosis G542X nonsensemutation suppression dose-response cell-free assays conducted forexemplary compounds according to some embodiments of the presentinvention, NB150, NB151 and NB152, at a concentration rage of 0-50 aM,wherein FIG. 7A shows the expression level of the firefly luciferase,which is found downstream of the mutant sequence, as a fraction of theexpression level exhibited in the control experiment (no addedcompound), and FIG. 7B shows the firefly/renilla expression ratio, downand upstream of the mutant sequence, as a fraction of the expressionlevel in the control experiment;

FIGS. 8A-C present the results of Rett syndrome R168X (FIG. 8A), R270X(FIG. 8B) and R294X (FIG. 8C) nonsense mutations suppression cell-freeassays conducted for exemplary compounds according to some embodimentsof the present invention, NB144, NB145, NB146, NB150, NB151 and NB152,at a concentration of 5 μM, showing the firefly/renilla expression ratioas a fraction of the firefly/renilla expression ratio measured for thecontrol sample (no compound added; 100%);

FIGS. 9A-B present ¹H NMR magnetic anisotropy spectra of Compound 35(upper spectrum) and Compound 36 (lower spectrum), showing thedifference in chemical shift values for the assigned protons in the NMRspectra (FIG. 9A), and the corresponding MαNP Sector Rule (FIG. 9B).

FIG. 10 presents comparative plots showing in vitro stop codonsuppression levels induced by Compound 1 (-▪-), NB153 (-▴-), and NB155(-Δ-) in R3X nonsense mutation construct representing USH1 geneticdisease.

FIGS. 11A-D presents comparative plots showing in vitro stop codonsuppression levels induced by NB74 (-Δ-), NB156 (-▴-), and gentamicin(-▪-) (left) and by NB124 (-Δ-), NB157 (-▴-), and gentamicin (-▪-)(right), in nonsense constructs representing R3X (USH1) (FIG. 11A),R245X (USH1) (FIG. 11B), Q70X (HS) (FIG. 11C), and G542X (CF) (FIG.11D). FIG.

FIG. 12A presents comparative stop-codon mutation readthrough plots,showing percent readthrough as a function of concentration of WT withNB156 (readthrough to 50% renilla), comparing the readthrough of severaldifferent mutations;

FIG. 12B presents comparative stop-codon mutation readthrough plots,showing fold increase of readthrough after exposure to NB156 fromnon-treated control as a function of NB156 concentration, comparing thereadthrough of several different mutations;

FIG. 13A presents comparative stop-codon mutation readthrough plots,showing percent readthrough as a function of concentration of WT withNB157 (readthrough to 50% renilla), comparing the readthrough of severaldifferent mutations; and

FIG. 13B presents comparative stop-codon mutation readthrough plots,showing fold increase of readthrough after exposure to NB157 fromnon-treated control as a function of NB157 concentration, comparing thereadthrough of several different mutations.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates toaminoglycosides and more particularly, but not exclusively, to novelaminoglycoside derivatives and their use in increasing an expression ofa gene having a stop codon mutation and/or in the treatment of geneticdisorders.

Specifically, the present invention, in some embodiments thereof,relates to a novel aminoglycoside compounds, derived from paromomycin,which exhibit high premature stop codon mutations readthrough activitywhile exerting low toxicity in mammalian cells, and which arecharacterized by improved bioavailability and/or cell permeability.Embodiments of the present invention are further of pharmaceuticalcompositions containing these compounds, and of uses thereof in thetreatment of genetic disorders. Embodiments of the present invention arefurther of processes of preparing these compounds.

The principles and operation of the present invention may be betterunderstood with reference to the figures and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or exemplified bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

As discussed hereinabove, the use aminoglycosides as therapeutic agentsis limited primarily due to their high toxicity. In the context oftreatment of genetic disorders, such a use is further limited by theantibacterial activity exhibited by the aminoglycosides, which can alsotranslate into toxicity.

Additional limitations associated with aminoglycosides include lowbioavailability, which typically requires an intravenous or subcutaneousadministration, and poor permeability into eukaryotic cells, whichtypically requires administration of high doses which are associatedwith adverse side effected. It is assumed that the high water solubilityand polarity of aminoglycosides limits their absorbance throughintestinal tissues and their permeability through cell membranes.

As further discussed hereinabove, several structural manipulations onthe structure of paromamine have given rise to synthetic aminoglycosideswhich have been shown to exert improved premature stop codon mutationsreadthrough activity while exerting low toxicity in mammalian cells. WO2007/113841 and WO 2012/066546, which are incorporated by reference asif fully set forth herein, describe such aminoglycosides.

While further deciphering the structure-activity relationship of suchaminoglycosides, in an attempt to further improve their therapeuticeffect in the context of genetic disorders, the present inventor hasdesigned numerous additional modifications, at varying positions of theparomamine structure, which are collectively represented herein byFormulae I and Ia.

While reducing the present invention to practice, exemplary novelaminoglycosides structures were prepared. As demonstrated in theExamples section that follows, these compounds were sown to exhibit highreadthrough activity of disease-causing nonsense mutations as well asreduced toxicity.

The Compounds:

According to an aspect of some embodiments of the present invention,there are provided novel aminoglycoside (AMG) compounds (also referredto herein as “aminoglycoside derivatives”), which are collectivelyrepresented by Formula Ia:

wherein:

the dashed line indicates a stereo-configuration of position 6′ being anR configuration or an S configuration;

X₁ is O or S;

the dashed bond between C4′ and C5′ in Ring I represents a single bondor a double bond;

the dashed bond between C4′ and C3′ in Ring I represents a single bondor a double bond;

Rx, Ry1 and Rz are each independently selected from hydrogen, alkyl,alkenyl, alkynyl, alkaryl, aryl heteroaryl and cycloalkyl, or absent,wherein at least Rz is absent in case the dashed bond between C4′ andC5′ is a double bond, and at least Ry1 is absent in case the dashed bondbetween C4′ and C3′ is a double bond;

Ry2-Ry9 and Rw1-Rw3 are each independently selected from hydrogen,alkyl, alkenyl, alkynyl, alkaryl, aryl, heteroaryl and cycloalkyl, eachbeing substituted or unsubstituted, or, alternatively, each can be asdefined herein for R₇-R₉;

R₁ is selected from the group consisting of hydrogen, a substituted orunsubstituted alkyl, a substituted or unsubstituted alkenyl, asubstituted or unsubstituted alkynyl, a substituted or unsubstitutedcycloalkyl, a substituted or unsubstituted aryl, a substituted orunsubstituted heteroaryl, a substituted or unsubstituted alkaryl, asubstituted or unsubstituted amine, a substituted or unsubstitutedamide, an acyl, a carboxylate, and a saturated or unsaturated and/or asubstituted or unsubstituted hydroxy alkyl (e.g., —CH₂—OH);

R₂ is selected from the group consisting of hydrogen, a substituted orunsubstituted alkyl, a substituted or unsubstituted alkenyl, asubstituted or unsubstituted alkynyl, a substituted or unsubstitutedcycloalkyl, a substituted or unsubstituted aryl, a substituted orunsubstituted heteroaryl, a substituted or unsubstituted alkaryl andacyl;

R₃ and R₄ are each independently selected from the group consisting ofhydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heteroalicyclic, aryl,heteroaryl, amine and OR₁₆, wherein R₁₆ is independently (when 2 or moreof R₃-R₆ is said OR₁₆) selected from hydrogen, a monosaccharide moiety,an oligosaccharide moiety, a substituted or unsubstituted alkyl, asubstituted or unsubstituted alkenyl, a substituted or unsubstitutedalkynyl, a substituted or unsubstituted cycloalkyl, a substituted orunsubstituted aryl, a substituted or unsubstituted heteroaryl, asubstituted or unsubstituted alkaryl and acyl, or is absent, wherein R₃is optionally absent in case the dashed bond between C4′ and C5′ is adouble bond, and R₄ is optionally absent in case the dashed bond betweenC4′ and C3′ is a double bond; and

R₇-R₉ are each independently selected from the group consisting ofhydrogen, acyl, an amino-substituted alpha-hydroxy acyl, a substitutedor unsubstituted alkyl, a substituted or unsubstituted alkenyl, asubstituted or unsubstituted alkynyl, a substituted or unsubstitutedcycloalkyl, a substituted or unsubstituted aryl, a substituted orunsubstituted alkaryl, carboxylate, sulfonyl (including alkyl sulfonyland aryl sulfonyl) and a cell-permealizable group.

In some of any of the embodiments described herein, the compound is apseudo-disaccharide, having Ring I and Ring II as depicted in FormulaIa.

In these embodiments, none of R₃-R₆ is OR₁₆ in which R₁₆ is amonosaccharide or an oligosaccharide moiety.

In some of these embodiments, one or more, or all, of R₃-R₆ is OR₁₆.

In some of these embodiments, one or more, or all, of R₃-R₆ is OR₁₆ andR₁₆ is independently an aryl, which can be substituted or unsubstituted.In these embodiments, one or more, or all, of R₃-R₆ is an aryloxy, asdefined herein.

In some of these embodiments, the aryl is unsubstituted such that one ormore, or all of R₃-R₆, independently, can be, as non-limiting examples,phenyloxy, 1-anthryloxy, 1-naphthyloxy, 2-naphthyloxy, 2-phenanthryloxyand 9-phenanthryloxy.

In some of these embodiments, one or more of the aryls in one or more ofOR₁₆ is a substituted aryl, such that one or more, or all of R₃-R₆,independently, can be, as non-limiting examples, an aryloxy in which thearyl is 2-(N-ethylamino)phenyl, 2-(N-hexylamino)phenyl,2-(N-methylamino)phenyl, 2,4-dimethoxyphenyl, 2-acetamidophenyl,2-aminophenyl, 2-carboxyphenyl, 2-chlorophenyl, 2-ethoxyphenyl,2-fluorophenyl, 2-hydroxymethylphenyl, 2-hydroxyphenyl, 2-hydroxyphenyl,2-methoxycarbonylphenyl, 2-methoxyphenyl, 2-methylphenyl,2-N,N-dimethylaminophenyl, 2-trifluoromethylphenyl,3-(N,N-dibutylamino)phenyl, 3-(N,N-diethylamino)phenyl,3,4,5-trimethoxyphenyl, 3,4-dichlorophenyl, 3,4-dimethoxyphenyl,3,5-dimethoxyphenyl, 3-aminophenyl, 3-biphenylyl, 3-carboxyphenyl,3-chloro-4-methoxyphenyl, 3-chlorophenyl, 3-ethoxycarbonylphenyl,3-ethoxyphenyl, 3-fluorophenyl, 3-hydroxymethylphenyl, 3-hydroxyphenyl,3-isoamyloxyphenyl, 3-isobutoxyphenyl, 3-isopropoxyphenyl,3-methoxyphenyl, 3-methylphenyl, 3-N,N-dimethylaminophenyl, 3-tolyl,3-trifluoromethylphenyl, 4-(benzyloxy)phenyl,4-(isopropoxycarbonyl)phenyl, 4-(N,N-diethylamino)phenyl,4-(N,N-dihexylamino)phenyl, 4-(N,N-diisopropylamino)phenyl,4-(N,N-dimethylamino)phenyl, 4-(N,N-di-n-pentylamino)phenyl,4-(n-hexyloxycarbonyl)phenyl, 4-(N-methylamino)phenyl,4-(trifluoromethyl)phenyl, 4-aminophenyl, 4-benzyloxyphenyl,4-biphenylyl, 4-butoxyphenyl, 4-butyramidophenyl, 4-carboxyphenyl,4-chlorophenyl, 4-ethoxycarbonylphenyl, 4-hexanamidophenyl,4-hydroxymethylphenyl, 4-hydroxyphenyl, 4-iodophenyl, 4-isobutylphenyl,4-isobutyramidophenyl, 4-isopropoxyphenyl, 4-isopropylphenyl,4-methoxyphenyl, 4-methylphenyl, 4-n-hexanamidophenyl,4-n-hexyloxyphenyl, 4-n-hexylphenyl, 4-nitrophenyl, 4-nitrophenyl,4-propionamidophenyl, 4-tolyl, 4-trifluoromethylphenyl and/or4-valeroyloxycarbonylphenyl.

In some of these embodiments, one or more, or all, of R₃-R₆ is OR₁₆ andR₁₆ is independently a heteroaryl, which can be substituted orunsubstituted. In these embodiments, one or more, or all, of R₃-R₆ is aheteroaryloxy, as defined herein.

In some embodiments, one or more, or all of R₃-R₆, independently, canbe, as non-limiting examples, 2-anthryloxy, 2-furyloxy, 2-indolyloxy,2-naphthyloxy, 2-pyridyloxy, 2-pyrimidyloxy, 2-pyrryloxy, 2-quinolyloxy,2-thienyloxy, 3-furyloxy, 3-indolyloxy, 3-thienyloxy, 4-imidazolyloxy,4-pyridyloxy, 4-pyrimidyloxy, 4-quinolyloxy, 5-methyl-2-thienyloxy and6-chloro-3-pyridyloxy.

In some of any of the embodiments described herein, R₃ is aryloxy orheteroaryloxy, as described herein.

In some of any of the embodiments described herein, R₃ is OR₁₆ and R₁₆is a substituted or unsubstituted alkyl or alkenyl, for example, methyl,ethyl, propyl, butyl, pentyl, propenyl, 2-hydroxyethyl, 3-hydroxypropyl,2,3-dihydroxypropyl and methoxymethyl.

In some of any of the embodiments described herein, R₃ is OR₁₆ and R₁₆is hydrogen.

In some of any of the embodiments described herein, R₄ is OR₁₆ and R₁₆is hydrogen.

In some of any of the embodiments described herein, each of R₃ and R₄ isOR₁₆ and R₁₆ is hydrogen.

In some of any of the embodiments described herein, one or more of, orall, of R₃-R₆ are OR₁₆.

In some of these embodiments, in each of R₃-R₆, R₁₆ is hydrogen.

In some of these embodiments, in one or more, or all, of R₃-R₆, R₁₆ isother than hydrogen.

In some of any of the embodiments described herein, when one or more, orall, of R₃-R₆ is OR₁₆ and when one or more, or all, of the R₁₆ moiety isother than hydrogen, R₁₆ can be the same or different for each of R₃-R₆.

In some of these embodiments, when in one or more, or all, of R₃-R₆, R₁₆is other than hydrogen, R₁₆ can be, for example, independently, alkyl,alkenyl, alkynyl, cycloalkyl, aryl or heteroaryl, each being optionallysubstituted, as described herein.

In some of any of the embodiments described herein, in one or more, orall, of R₃-R₆, R₁₆ is independently an acyl, forming an ester (acarboxylate) at the respective position.

Herein throughout, the term “acyl” describes a —C(═O)—R′ group, whereinR′ is as described herein.

In some of any of the embodiments described herein, In some of any ofthe embodiments described herein, when R₁₆ is an acyl, R′ is ahydrocarbon chain, as described herein, optionally substituted. In someembodiments, the hydrocarbon chain is of 2 to 18 carbon atoms in length.In some embodiments, the acyl is a hydrocarbon acyl radical having from2 to 18 carbon atoms, optionally substituted by one or more of halo,nitro, hydroxy, amine, cyano, thiocyano, and alkoxy.

Herein, the term “hydrocarbon” or “hydrocarbon radical” describes anorganic moiety that includes, as its basic skeleton, a chain of carbonatoms, also referred to herein as a backbone chain, substituted mainlyby hydrogen atoms. The hydrocarbon can be saturated or non-saturated, becomprised of aliphatic, alicyclic and/or aromatic moieties, and canoptionally be substituted by one or more substituents (other thanhydrogen). A substituted hydrocarbon may have one or more substituents,whereby each substituent group can independently be, for example, alkyl,cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, amine,halide, sulfonate, sulfoxide, phosphonate, hydroxy, alkoxy, aryloxy,thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, azo, azide,sulfonamide, carboxy, thiocarbamate, urea, thiourea, carbamate, amide,and hydrazine, and any other substituents as described herein.

The hydrocarbon moiety can optionally be interrupted by one or moreheteroatoms, including, without limitation, one or more oxygen, nitrogen(substituted or unsubstituted, as defined herein for —NR′—) and/orsulfur atoms.

In some embodiments of any of the embodiments described herein relatingto a hydrocarbon, the hydrocarbon is not interrupted by any heteroatom,nor does it comprise heteroatoms in its backbone chain, and can be analkylene chain, or be comprised of alkyls, cycloalkyls, aryls, alkenesand/or alkynes, covalently attached to one another in any order.

In some of any of the embodiments described herein, when R₁₆ is an acyl,the acyl can be derived from a carboxylic acid, such that the esterformed at the respective position is derived from, for example, asaturated or unsaturated and/or substituted or unsubstituted aliphaticcarboxylic acid, including, but not limited to, acetic acid, propionicacid, butyric acid, isobutyric acid, tert-butylacetic acid, valericacid, isovaleric acid, caproic acid, caprylic acid, decanoic acid,dodecanoic acid, lauric acid, tridecanoic acid, myristic acid,pentadecanoic acid, palmitic acid, margaric acid, stearic acid, acrylicacid, crotonic acid, undecylenic acid, oleic acid, hexynoic acid,heptynoic acid, octynoic acid; a saturated or unsaturated alicycliccarboxylic acid, including, but not limited to, cyclobutanecarboxylicacid, cyclopentanecarboxylic acid, cyclopentenecarboxylic acid,methylcyclopentenecarboxylic acid, cyclohexanecarboxylic acid,dimethylcyclohexanecarboxylic acid, dipropylcyclohexanecarboxylic acid;a saturated or unsaturated, alicyclic aliphatic carboxylic acid,including, but not limited to, cyclopentaneacetic acid,cyclopentanepropionic acid, cyclohexaneacetic acid, cyclohexanebutyricacid, methylcyclohexaneacetic acid, a substituted or unsubstitutedaromatic carboxylic acid, benzoic acid, toluic acid, naphthoic acid,ethylbenzoic acid, isobutylbenzoic acid, methylbutylbenzoic acid; anaromatic carboxylic acid, including, but not limited to, phenylaceticacid, benzoic acid, phenylpropionic acid, phenylvaleric acid, cinnamicacid, phenylpropiolic acid, naphthylacetic acid; ahalo-alkoxyhydrocarbon carboxylic acid; a nitro-alkoxyhydrocarboncarboxylic acid; a hydroxy-alkoxyhydrocarbon carboxylic acid; anamino-alkoxyhydrocarbon carboxylic acid; a cyano-alkoxyhydrocarboncarboxylic acid; a thiocyano-alkoxyhydrocarbon carboxylic acid; as wellas mono-acetic acid; di-acetic acid, trichloroacetic acid;1,2,3,4,5,6-hexachlorocyclohexanecarboxylic acid,1,2-dibromo-4-methylcyclohexanecarboxylic acid,1,6-dibromo-3-methylcyclohexanecarboxylic acid,1-bromo-3,5-dimethylcyclohexanecarboxylic acid,2-chlorocyclohexanecarboxylic acid, 4-chlorocyclohexanecarboxylic acid,2,3-dibromo-2-methylcyclohexanecarboxylic acid, 2,4,6-trinitrobenzoicacid, 2,5-dibromo-2-methylcyclohexanecarboxylic acid,2-bromo-4-methylcyclohexanecarboxylic acid,2-nitro-1-methyl-cyclobutanecarboxylic acid, 3,4-dinitrobenzoic acid,3,5-dinitrobenzoic acid, 3-bromo-2,2,3-trimethylcyclopentanecarboxylicacid, 3-bromo-2-methylcyclohexanecarboxylic acid,3-bromo-3-methylcyclohexanecarboxylic acid,4-bromo-2-methylcyclohexanecarboxylic acid,5-bromo-2-methylcyclohexanecarboxylic acid, ‘4,4-dichlorobenzilic acid,4,5-dibromo-2-methylcyclohexanecarboxylic acid,5-bromo-2-methylcyclohexanecarboxylic acid,6-bromo-2-methylcyclohexanecarboxylic acid,5,6-dibromo-2-methylcyclohexanecarboxylic acid,6-bromo-3-methylcyclohexanecarboxylic acid, anisic acid, cyanoaceticacid, cyanopropionic acid, ethoxyformic acid (ethyl hydrogen carbonate),gallic acid, homogentisic acid, o-, m-, and p-chlorobenzoic acid, lacticacid, mevalonic acid, o-, m-, p-nitrobenzoic acid, p-hydroxybenzoicacid, salicylic acid, shikimic acid, thiocyanoacetic acid,trimethoxybenzoic acid, trimethoxycinnamic acid, veratric acid, α- andβ-chloropropionic acid, α- and γ-bromobutyric acid and α- andδ-iodovaleric acid, β-resorcylic acid.

In some of any of the embodiments described herein, when one or more ofR₇-R₉ is acyl, the acyl is such that R′ is an alkyl or alkaryl or aryl,each of which being optionally substituted by one or more aminesubstituents.

In some embodiments, R is a substituted alkyl, and in some embodiments,R is substituted by hydroxy at the α position with respect to thecarbonyl group, such that the acyl is α-hydroxy-acyl.

In some embodiments, the α-hydroxy-acyl is further substituted by one ormore amine groups, and is an amino-substituted α-hydroxy-acyl.

In some of the embodiments of an acyl group as described herein, theamine substituents can be, for example, at one or more of positions β,γ, δ, and/or ω of the moiety R, with respect to the acyl.

Exemplary amino-substituted α-hydroxy-acyls include, without limitation,the moiety (S)-4-amino-2-hydroxybutyryl, which is also referred toherein as AHB. According to some embodiments of the present invention,an alternative to the AHB moiety can be the α-hydroxy-β-aminopropionyl(AHP) moiety. Additional exemplary amino-substituted α-hydroxy-acylsinclude, but are not limited to, L-(−)-γ-amino-α-hydroxybutyryl,L(−)-δ-amino-α-hydroxyvaleryl,L-(−)-β-benzyloxycarbonylamino-α-hydroxypropionyl, aL-(−)-δ-benzyloxycarbonylamino-α-hydroxyvaleryl

It is noted herein that according to some embodiments of the presentinvention, other moieties which involve a combination of carbonyl(s),hydroxyl(s) and amino group(s) along a lower alkyl exhibiting anystereochemistry, are contemplated as optional substituents in place ofAHB and/or AHP, including, for example, 2-amino-3-hydroxybutanoyl,3-amino-2-hydroxypentanoyl, 5-amino-3-hydroxyhexanoyl and the likes.

In some of any of the embodiments described herein, one or more of R₃-R₆is other than OR₁₆. In some of any of the embodiments described herein,one or more of R₃-R₆ is hydrogen.

In some of any of the embodiments described herein R₃ is hydrogen.

In some of any of the embodiments described herein R₄ is hydrogen.

In some of any of the embodiments described herein R₃ and R₄ are eachhydrogen.

In some of any of the embodiments described herein, one or more of R₃-R₆is OR₁₆ and R₁₆ is independently a monosaccharide moiety or anoligosaccharide moiety, as defined herein, such that the compound is apseudo-trisaccharide, a pseudo-tetrasaccharide, apseudo-pentasaccharide, a pseudo hexasaccharide, etc.

Whenever one or more of R₃-R₆ is OR₁₆ and R₁₆ is a monosaccharide moietyor an oligosaccharide moiety and one or more of R₃-R₆ is not OR₁₆ inwhich R₁₆ is a monosaccharide moiety or an oligosaccharide moiety, theone or more of R₃-R₆ is not OR₁₆ in which R₁₆ is a monosaccharide moietyor an oligosaccharide moiety can be as described herein for any of therespective embodiments for R₃-R₆.

The term “monosaccharide”, as used herein and is well known in the art,refers to a simple form of a sugar that consists of a single saccharidemolecule which cannot be further decomposed by hydrolysis. Most commonexamples of monosaccharides include glucose (dextrose), fructose,galactose, and ribose. Monosaccharides can be classified according tothe number of carbon atoms of the carbohydrate, i.e., triose, having 3carbon atoms such as glyceraldehyde and dihydroxyacetone; tetrose,having 4 carbon atoms such as erythrose, threose and erythrulose;pentose, having 5 carbon atoms such as arabinose, lyxose, ribose,xylose, ribulose and xylulose; hexose, having 6 carbon atoms such asallose, altrose, galactose, glucose, gulose, idose, mannose, talose,fructose, psicose, sorbose and tagatose; heptose, having 7 carbon atomssuch as mannoheptulose, sedoheptulose; octose, having 8 carbon atomssuch as 2-keto-3-deoxy-manno-octonate; nonose, having 9 carbon atomssuch as sialose; and decose, having 10 carbon atoms. Monosaccharides arethe building blocks of oligosaccharides like sucrose (common sugar) andother polysaccharides (such as cellulose and starch).

The term “oligosaccharide” as used herein refers to a compound thatcomprises two or more monosaccharide units, as these are defined herein,linked to one another via a glycosyl bond (—O—). Preferably, theoligosaccharide comprises 2-6 monosaccharides, more preferably theoligosaccharide comprises 2-4 monosaccharides and most preferably theoligosaccharide is a disaccharide moiety, having two monosaccharideunits.

In some of any of the embodiments described herein, the monosaccharideis a pentose moiety, such as, for example, represented by Formula II.Alternatively, the monosaccharide moiety is hexose. Furtheralternatively, the monosaccharide moiety is other than pentose orhexose, for example, a hexose moiety as described in U.S. Pat. No.3,897,412.

In some of any of the embodiments described herein, the monosaccharidemoiety is a ribose, represented by Formula II:

wherein:

the curved line denotes a position of attachment;

the dashed line indicates a stereo-configuration of position 5″ being anR configuration or an S configuration;

X₂ is OR₁₃ or NR₁₄R₁₅;

each of R₁₀, R₁₁ and R₁₃ is independently selected from the groupconsisting of hydrogen, a substituted or unsubstituted alkyl, asubstituted or unsubstituted alkenyl, a substituted or unsubstitutedalkynyl, a substituted or unsubstituted cycloalkyl, a substituted orunsubstituted aryl, a substituted or unsubstituted heteroaryl, asubstituted or unsubstituted alkaryl, and acyl;

R₁₂ is selected from the group consisting of hydrogen, a substituted orunsubstituted alkyl, a substituted or unsubstituted alkenyl, asubstituted or unsubstituted alkynyl, a substituted or unsubstitutedcycloalkyl, a substituted or unsubstituted aryl, a substituted orunsubstituted heteroaryl, a substituted or unsubstituted alkaryl, asubstituted or unsubstituted amine, a substituted or unsubstitutedamide, an acyl, a carboxylate, and a saturated or unsaturated and/orsubstituted or unsubstituted hydroxyalkyl;

each of R₁₄ and R₁₅ is independently selected from the group consistingof hydrogen, a substituted or unsubstituted alkyl, a substituted orunsubstituted alkenyl, a substituted or unsubstituted alkynyl, asubstituted or unsubstituted cycloalkyl, a substituted or unsubstitutedaryl, a substituted or unsubstituted heteroaryl, a substituted orunsubstituted alkaryl, acyl, and a cell-permealizable group, or,alternatively, R₁₄ and R₁₅, when present, form together a heterocyclicring.

In some embodiments, X₂ is OR₁₃.

In some embodiments, X₂ is NR₁₄R₁₅.

In some of any of the embodiments described herein, R₁₂ is other thanhydrogen.

In some of these embodiments, R₁₂ is alkyl, cycloalkyl or aryl, and insome embodiments, R₁₂ is alkyl, preferably a lower alkyl, for example,methyl.

In some embodiments, R₁₂ is as defined herein for R₁.

In some of any of the embodiments where one or more of R₃-R₆ is OR₁₆ andR₁₆ is a monosaccharide moiety or an oligosaccharide moiety, one or moreof the hydroxy groups in the monosaccharide or oligosaccharidemoiety/moieties are substituted by an acyl, forming an ester (acarboxylate), as described herein in any of the respective embodiments.

In some of any of the embodiments described herein, one of R₃-R₆ is OR₁₆and R₁₆ is a monosaccharide moiety such that the compound is apseudo-trisaccharide.

In some of any of the embodiments described herein for apseudo-trisaccharide, one or more, or all, of R₁₀ and R₁₁, and R₁₃ ifpresent, can be an acyl, as described herein.

In some of any of the embodiments described herein for apseudo-trisaccharide, one or more, or all, of R₃-R₆ are OR₁₆, such thatin one of R₃-R₆, R₁₆ is a monosaccharide moiety, and in the others, R₁₆is as defined herein (e.g., hydrogen, acyl).

In some of any of the embodiments described herein, R₅ is OR₁₆ in whichR₁₆ is a monosaccharide moiety.

In some of these embodiments, the compound is represented by Formula Ib:

with the variables being as described herein for Formulae Ia and II,including any combination thereof.

In some of any of the embodiments described herein for Formulae Ia andIb, X₁ is O.

In some of any of the embodiments described herein, the bond between C4′and C5′ in Ring I is a single bond.

In some of any of the embodiments described herein, the bond between C4′and C5′ in Ring I is a double bond. In some of these embodiments, Rx andRz are absent. Alternatively, R₃ and Rz are absent.

In some of any of the embodiments described herein, the bond between C4′and C3′ in Ring I is a single bond.

In some of any of the embodiments described herein, the bond between C4′and C3′ in Ring I is a double bond. In some of these embodiments, Rx andRy1 are absent. Alternatively, R₄ and Ry1 are absent.

In some of any of the embodiments described herein, one or more, or all,of Rx, Rz, Ry1, if present, and Ry2-Ry9 and Rw1-Rw3 is/are hydrogen.

In some of any of the embodiments described herein, R₁ is other thanhydrogen.

In some of any of the embodiments described herein, R₁ is ahydroxyalkyl, wherein the alkyl can be further substituted or not.

In some of any of the embodiments described herein, R₁ is ahydroxymethyl.

In some of any of the embodiments described herein, R₁ is alkyl, alkenylor alkynyl, each being substituted or unsubstituted.

In some of any of the embodiments described herein, R₁ is alkyl,preferably a lower alkyl, for example, methyl, ethyl, propyl, butyl orpentyl.

In some of any of the embodiments described herein, R₁ is or comprisesan aryl which can be substituted or unsubstituted. In some embodiments,R₁ is an unsubstituted aryl and can be, as non-limiting examples,phenyl, 1-anthryl, 1-naphthyl, 2-naphthyl, 2-phenanthryl or9-phenanthryl.

In some embodiments, R₁ is a substituted aryl, and can be, asnon-limiting examples, 2-(N-ethylamino)phenyl, 2-(N-hexylamino)phenyl,2-(N-methylamino)phenyl, 2,4-dimethoxyphenyl, 2-acetamidophenyl,2-aminophenyl, 2-carboxyphenyl, 2-chlorophenyl, 2-ethoxyphenyl,2-fluorophenyl, 2-hydroxymethylphenyl, 2-hydroxyphenyl, 2-hydroxyphenyl,2-methoxycarbonylphenyl, 2-methoxyphenyl, 2-methylphenyl,2-N,N-dimethylaminophenyl, 2-trifluoromethylphenyl,3-(N,N-dibutylamino)phenyl, 3-(N,N-diethylamino)phenyl,3,4,5-trimethoxyphenyl, 3,4-dichlorophenyl, 3,4-dimethoxyphenyl,3,5-dimethoxyphenyl, 3-aminophenyl, 3-biphenylyl, 3-carboxyphenyl,3-chloro-4-methoxyphenyl, 3-chlorophenyl, 3-ethoxycarbonylphenyl,3-ethoxyphenyl, 3-fluorophenyl, 3-hydroxymethylphenyl, 3-hydroxyphenyl,3-isoamyloxyphenyl, 3-isobutoxyphenyl, 3-isopropoxyphenyl,3-methoxyphenyl, 3-methylphenyl, 3-N,N-dimethylaminophenyl, 3-tolyl,3-trifluoromethylphenyl, 4-(benzyloxy)phenyl,4-(isopropoxycarbonyl)phenyl, 4-(N,N-diethylamino)phenyl,4-(N,N-dihexylamino)phenyl, 4-(N,N-diisopropylamino)phenyl,4-(N,N-dimethylamino)phenyl, 4-(N,N-di-n-pentylamino)phenyl,4-(n-hexyloxycarbonyl)phenyl, 4-(N-methylamino)phenyl,4-(trifluoromethyl)phenyl, 4-aminophenyl, 4-benzyloxyphenyl,4-biphenylyl, 4-butoxyphenyl, 4-butyramidophenyl, 4-carboxyphenyl,4-chlorophenyl, 4-ethoxycarbonylphenyl, 4-hexanamidophenyl,4-hydroxymethylphenyl, 4-hydroxyphenyl, 4-iodophenyl, 4-isobutylphenyl,4-isobutyramidophenyl, 4-isopropoxyphenyl, 4-isopropylphenyl,4-methoxyphenyl, 4-methylphenyl, 4-n-hexanamidophenyl,4-n-hexyloxyphenyl, 4-n-hexylphenyl, 4-nitrophenyl, 4-nitrophenyl,4-propionamidophenyl, 4-tolyl, 4-trifluoromethylphenyl or4-valeroyloxycarbonylphenyl.

In some of any of the embodiments described herein, R₁ is or comprises asubstituted or unsubstituted heteroaryl, and can be, as non-limitingexamples, 2-anthryl, 2-furyl, 2-indolyl, 2-naphthyl, 2-pyridyl,2-pyrimidyl, 2-pyrryl, 2-quinolyl, 2-thienyl, 3-furyl, 3-indolyl,3-thienyl, 4-imidazolyl, 4-pyridyl, 4-pyrimidyl, 4-quinolyl,5-methyl-2-thienyl and 6-chloro-3-pyridyl.

In some of any of the embodiments described herein, R₁ is or comprisesan amine, as defined herein, and can be, as non-limiting examples, —NH₂,—NHCH₃, —N(CH₃)₂, —NH—CH₂—CH₂—NH₂, —NH—CH₂—CH₂—OH and —NH—CH₂—CH(OCH₃)₂.

In some of any of the embodiments described herein, R₁ is alkyl, and insome embodiments it is a lower alkyl, of 1 to 4 carbon atoms, including,but not limited to, methyl, ethyl, propyl, butyl, isopropyl, andisobutyl.

In some of any of the embodiments described herein, R₁ is anon-substituted alkyl.

In some of any of the embodiments described herein, R₁ is methyl.

Alternatively, in some of any of the embodiments described herein, R₁ iscycloalkyl, including, but not limited to, cyclopropyl, cyclobutyl,cyclopentyl and cyclohexyl.

Further alternatively, in some of any of the embodiments describedherein, R₁ is aryl, such as substituted or unsubstituted phenyl.Non-limiting examples include unsubstituted phenyl and toluene.

Further alternatively, in some of any of the embodiments describedherein, R₁ is alkaryl, such as, for example, a substituted orunsubstituted benzyl.

In some of any of the embodiments described herein, R₁ is other thanalkyl, cycloalkyl and aryl.

In some of any of the embodiments described herein, R₁ is other thanalkyl, cycloalkyl and aryl, wherein each is unsubstituted.

In some of any of the embodiments described herein, R₁ is other thanmethyl.

In some of any of the embodiments described herein, R₂ is hydrogen.

In some of any of the embodiments described herein, R₂ is other thanhydrogen.

In some of any of the embodiments described herein, R₂ is an acyl,forming as ester at this position, as described herein.

In some embodiments, R₂ is alkyl, preferably selected from the groupconsisting of methyl, ethyl and propyl.

In some of any of the embodiments described herein, R₂ is alkyl, and insome of these embodiments R₂ is a substituted alkyl, for example, analkyl substituted by one or more amine groups (aminoalkyl).

In some of any of the embodiments described herein, R₂ is a substitutedor unsubstituted alkyl, as defined herein, or a substituted orunsubstituted cycloalkyl, as defined herein.

In some of any of the embodiments described herein, R₂ is a substitutedor unsubstituted aryl, as defined herein.

In some of any of the embodiments described herein, R₁ is hydroxyalkyland R₂ is hydrogen.

In some of any of the embodiments described herein, R₁ is hydroxyalkyland R₂ is an acyl.

In some of any of the embodiments described herein, one or more of R₇-R₉and of R₁₄ and R₁₅, if present, is independently an alkyl, acell-permealizable group, as described herein, or an acyl, such as, forexample, an alpha-hydroxy acyl or an amino-substituted alpha-hydroxyacyl, as described herein.

In some of any of the embodiments described herein, one or more of R₇-R₉and of R₁₄ and R₁₅, if present, is a sulfonyl, for example, an alkylsulfonyl or an aryl sulfonyl.

Exemplary moieties represented by one or more of R₇-R₉ and of R₁₄ andR₁₅, if present, include, but are not limited to, hydrogen,(R/S)-4-amino-2-hydroxybutyryl (AHB), (R/S)-3-amino-2-hydroxypropionyl(AHP), 5-aminopentanoyl, 5-hydroxypentanoyl, formyl, —C(═O)—O-methyl,—C(═O)—O-ethyl, —C(═O)—O-benzyl, -β-amino-α-hydroxypropionyl,-δ-amino-α-hydroxyvaleryl, -β-benzyloxycarbonylamino-α-hydroxypropionyl,-δ-benzyloxycarbonylamino-α-hydroxyvaleryl, methylsulfonyl,phenylsulfonyl, benzoyl, propyl, isopropyl, —(CH₂)₂NH₂, —(CH₂)₃NH₂,—CH₂CH(NH₂)CH₃, —(CH₂)₄NH₂, —(CH₂)₅NH₂, —(CH₂)₂NH-ethyl,—(CH₂)₂NH(CH₂)₂NH₂, —(CH₂)₃NH(CH₂)₃NH₂, —(CH₂)₃NH(CH₂)₄NH(CH₂)₃NH₂,—CH(—NH₂)CH₂(OH), —CH(—OH)CH₂(NH₂), —CH(—OH)—(CH₂)₂(NH₂),—CH(—NH₂)—(CH₂)₂(OH), —CH(—CH₂NH₂)—(CH₂OH), —(CH₂)₄NH(CH₂)₃NH₂,—(CH₂)₂NH(CH₂)₂NH(CH₂)₂NH₂, —(CH₂)₂N(CH₂CH₂NH₂)₂, —CH₂—C(═O)NH₂,—CH(CH₃)—C(═O)NH₂, —CH₂-phenyl, —CH(i-propyl)-C(═O)NH₂,—CH(benzyl)-C(═O)NH₂, —(CH₂)₂₀H, —(CH₂)₃OH and —CH(CH₂OH)₂.

In some of any of the embodiments described herein, R₇ is hydrogen,(R/S)-4-amino-2-hydroxybutyryl (AHB), (R/S)-3-amino-2-hydroxypropionyl,5-aminopentanoyl, 5-hydroxypentanoyl, formyl, —C(═O)—O-methyl,—C(═O)—O-ethyl, —C(═O)—O-benzyl, -β-amino-α-hydroxypropionyl,-δ-amino-α-hydroxyvaleryl, -β-benzyloxycarbonylamino-α-hydroxypropionyl,-δ-benzyloxycarbonylamino-α-hydroxyvaleryl, methylsulfonyl,phenylsulfonyl, benzoyl, propyl, isopropyl, —(CH₂)₂NH₂, —(CH₂)₃NH₂,—CH₂CH(NH₂)CH₃, —(CH₂)₄NH₂, —(CH₂)₅NH₂, —(CH₂)₂NH-ethyl,—(CH₂)₂NH(CH₂)₂NH₂, —(CH₂)₃NH(CH₂)₃NH₂, —(CH₂)₃NH(CH₂)₄NH(CH₂)₃NH₂,—CH(—NH₂)CH₂(OH), —CH(—OH)CH₂(NH₂), —CH(—OH)—(CH₂)₂(NH₂),—CH(—NH₂)—(CH₂)₂(OH), —CH(—CH₂NH₂)—(CH₂OH), —(CH₂)₄NH(CH₂)₃NH₂,—(CH₂)₂NH(CH₂)₂NH(CH₂)₂NH₂, —(CH₂)₂N(CH₂CH₂NH₂)₂, —CH₂—C(═O)NH₂,—CH(CH₃)—C(═O)NH₂, —CH₂-phenyl, —CH(i-propyl)-C(═O)NH₂,—CH(benzyl)-C(═O)NH₂, —(CH₂)₂₀H, —(CH₂)₃OH or —CH(CH₂OH)₂.

In some of any of the embodiments described herein, R₇ is other thanhydrogen, (R/S)-4-amino-2-hydroxybutyryl (AHB), and(R/S)-3-amino-2-hydroxypropionyl (AHP).

In some of any of the embodiments described herein, R₇ is other thanhydrogen, and in some of these embodiments, R₇ is other than anamino-substituted alpha-hydroxy acyl, as defined herein.

In some of any of the embodiments described herein, R₇ is other thanalkyl, cycloalkyl, aryl and a cell-permealizable group, as describedherein.

In some of any of the embodiments described herein, one or both of R₈and R₉ is independently hydrogen, (R/S)-4-amino-2-hydroxybutyryl (AHB),(R/S)-3-amino-2-hydroxypropionate (AHP),(R/S)-3-amino-2-hydroxypropionyl, 5-aminopentanoyl, 5-hydroxypentanoyl,formyl, —COO-methyl, —COO-ethyl, —COO-benzyl,-β-amino-α-hydroxypropionyl, -δ-amino-α-hydroxyvaleryl,-β-benzyloxycarbonylamino-α-hydroxypropionyl,-δ-benzyloxycarbonylamino-α-hydroxyvaleryl, methylsulfonyl,phenylsulfonyl, benzoyl, propyl, isopropyl, —(CH₂)₂NH₂, —(CH₂)₃NH₂,—CH₂CH(NH₂)CH₃, —(CH₂)₄NH₂, —(CH₂)₅NH₂, —(CH₂)₂NH-ethyl,—(CH₂)₂NH(CH₂)₂NH₂, —(CH₂)₃NH(CH₂)₃NH₂, —(CH₂)₃NH(CH₂)₄NH(CH₂)₃NH₂,—CH(—NH₂)CH₂(OH), —CH(—OH)CH₂(NH₂), —CH(—OH)—(CH₂)₂(NH₂),—CH(—NH₂)—(CH₂)₂(OH), —CH(—CH₂NH₂)—(CH₂OH), —(CH₂)₄NH(CH₂)₃NH₂,—(CH₂)₂NH(CH₂)₂NH(CH₂)₂NH₂, —(CH₂)₂N(CH₂CH₂NH₂)₂, —CH₂—C(═O)NH₂,—CH(CH₃)—C(═O)NH₂, —CH₂-phenyl, —CH(i-propyl)-C(═O)NH₂,—CH(benzyl)-C(═O)NH₂, —(CH₂)₂₀H, —(CH₂)₃OH or —CH(CH₂OH)₂.

In some of any of the embodiments described herein, an amino-substitutedalpha-hydroxy acyl is (S)-4-amino-2-hydroxybutyryl (AHB).

In some of any of the embodiments described herein, each of R₇-R₉ isother than hydrogen, (R/S)-4-amino-2-hydroxybutyryl (AHB), and(R/S)-3-amino-2-hydroxypropionyl (AHP).

In some of any of the embodiments described herein, each of R₇-R₉ isother than hydrogen, and in some of these embodiments, each of R₇-R₉ isother than an amino-substituted alpha-hydroxy acyl, as defined herein.

In some of any of the embodiments described herein, each of R₇-R₉ isother than alkyl, cycloalkyl, aryl and a cell-permealizable group, asdescribed herein.

Herein throughout, an amine which bears a substituent other thanhydrogen is referred to herein as a “modified amine substituent” orsimply as a “modified amine”.

According to some embodiments of the present invention, one or both ofthe amine substituents at positions 1 (R₇), 2′ (R₈), 3 (R₉) or 5″ (R₁₄or R₁₅), if present, of the aminoglycoside structure represented byFormulae Ia and Ib, is modified to include a hydrophobic moiety such asalkyl, cycloalkyl, alkaryl and/or aryl, or a group which ispositively-charged at physiological pH and which can increase cellpermeability of the compound (also referred to herein interchangeably as“cell-permealizable group” or “cell-permealizing group”), such asguanine or guanidine groups, as defined herein, or, alternatively,hydrazine, hidrazide, thiohydrazide, urea and thiourea.

In some of any of the embodiments described herein, one or more R₇-R₉and R₁₄ and R₁₅, if present, is a cell-permealizable group as definedherein, and in some embodiments, it is a guanidyl, as defined herein.

In some of any of the embodiments described herein, one or more R₇-R₉and R₁₄ and R₁₅, if present, is a hydrophobic moiety such as alkyl,cycloalkyl, alkaryl and/or aryl.

In some of any of the embodiments described herein, none of R₇-R₉ andR₁₄ and R₁₅, if present, is a hydrophobic moiety such as alkyl,cycloalkyl, alkaryl and/or aryl.

In some of any of the embodiments described herein, none of R₇-R₉ andR₁₄ and R₁₅, if present, is a cell-permealizable group, as definedherein.

In some of any of the embodiments described herein, none of R₇-R₉ andR₁₄ and R₁₅, if present, is a modified amine as described herein.

In some of any of the embodiments described herein, one or more R₇-R₉and R₁₄ and R₁₅, if present, is an acyl, as defined herein, and in someof these embodiments, the acyl can independently be an amino-substitutedalpha-hydroxy acyl, as defined herein.

In some of any of the embodiments described herein, whenever a variableis defined as an unsubstituted aryl, the unsubstituted aryl can be, forexample, phenyl, 1-anthryl, 1-naphthyl, 2-naphthyl, 2-phenanthryl and/or9-phenanthryl.

In some of any of the embodiments described herein, whenever a variableis defined as a substituted or unsubstituted heteroaryl, the heteroarylcan be, for example, 2-anthryl, 2-furyl, 2-indolyl, 2-naphthyl,2-pyridyl, 2-pyrimidyl, 2-pyrryl, 2-quinolyl, 2-thienyl, 3-furyl,3-indolyl, 3-thienyl, 4-imidazolyl, 4-pyridyl, 4-pyrimidyl, 4-quinolyl,5-methyl-2-thienyl and/or 6-chloro-3-pyridyl.

In some of any of the embodiments described herein, whenever a variableis defined as a substituted aryl, the aryl can be, for example,2-(N-ethylamino)phenyl, 2-(N-hexylamino)phenyl, 2-(N-methylamino)phenyl,2,4-dimethoxyphenyl, 2-acetamidophenyl, 2-aminophenyl, 2-carboxyphenyl,2-chlorophenyl, 2-ethoxyphenyl, 2-fluorophenyl, 2-hydroxymethylphenyl,2-hydroxyphenyl, 2-hydroxyphenyl, 2-methoxycarbonylphenyl,2-methoxyphenyl, 2-methylphenyl, 2-N,N-dimethylaminophenyl,2-trifluoromethylphenyl, 3-(N,N-dibutylamino)phenyl,3-(N,N-diethylamino)phenyl, 3,4,5-trimethoxyphenyl, 3,4-dichlorophenyl,3,4-dimethoxyphenyl, 3,5-dimethoxyphenyl, 3-aminophenyl, 3-biphenylyl,3-carboxyphenyl, 3-chloro-4-methoxyphenyl, 3-chlorophenyl,3-ethoxycarbonylphenyl, 3-ethoxyphenyl, 3-fluorophenyl,3-hydroxymethylphenyl, 3-hydroxyphenyl, 3-isoamyloxyphenyl,3-isobutoxyphenyl, 3-isopropoxyphenyl, 3-methoxyphenyl, 3-methylphenyl,3-N,N-dimethylaminophenyl, 3-tolyl, 3-trifluoromethylphenyl,4-(benzyloxy)phenyl, 4-(isopropoxycarbonyl)phenyl,4-(N,N-diethylamino)phenyl, 4-(N,N-dihexylamino)phenyl,4-(N,N-diisopropylamino)phenyl, 4-(N,N-dimethylamino)phenyl,4-(N,N-di-n-pentylamino)phenyl, 4-(n-hexyloxycarbonyl)phenyl,4-(N-methylamino)phenyl, 4-(trifluoromethyl)phenyl, 4-aminophenyl,4-benzyloxyphenyl, 4-biphenylyl, 4-butoxyphenyl, 4-butyramidophenyl,4-carboxyphenyl, 4-chlorophenyl, 4-ethoxycarbonylphenyl,4-hexanamidophenyl, 4-hydroxymethylphenyl, 4-hydroxyphenyl,4-iodophenyl, 4-isobutylphenyl, 4-isobutyramidophenyl,4-isopropoxyphenyl, 4-isopropylphenyl, 4-methoxyphenyl, 4-methylphenyl,4-n-hexanamidophenyl, 4-n-hexyloxyphenyl, 4-n-hexylphenyl,4-nitrophenyl, 4-nitrophenyl, 4-propionamidophenyl, 4-tolyl,4-trifluoromethylphenyl and/or 4-valeroyloxycarbonylphenyl.

In some of any of the embodiments described herein, the aminesubstituent at position 1 (R₇, Ring II) in Formula Ia or Ib, is amodified amine, as described herein, such that R₇ is other thanhydrogen.

In some of these embodiments, R₇ can be alkyl, cycloalkyl, alkaryl,aryl, an acyl, or an amino-substituted α-hydroxy acyl, as definedherein, such as, for example, (S)-4-amino-2-hydroxybutyryl (AHB), or(S)-4-amino-2-hydroxypropionyl (AHP).

In some of the embodiments where R₇ is alkyl, the alkyl can be, forexample, a lower alkyl, of 1-4 carbon atoms, such as, but not limitedto, methyl, ethyl, propyl, butyl, isopropyl, and isobutyl, each beingoptionally substituted, as described herein.

In some of these embodiments, the alkyl is independently anon-substituted alkyl, such as, but not limited to, ethyl, propyl andisopropyl.

In some of these embodiments, the alkyl is independently a substitutedmethyl, such as, but not limited to, an alkaryl such as benzyl.

Alternatively, R₇ is cycloalkyl, and the cycloalkyl can be, for example,cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

Further alternatively, R₇ is aryl, and the aryl can be, for example, asubstituted or unsubstituted phenyl. Non-limiting examples includeunsubstituted phenyl and toluene.

In some of any of the embodiments described herein, R₇ is alkyl,cycloalkyl or aryl, as described herein.

In some of these embodiments, R₁ is alkyl, cycloalkyl or aryl, and ispreferably alkyl, as defined herein.

In some of these embodiments, R₁ is alkyl, cycloalkyl or aryl, and ispreferably alkyl, as defined herein, R₃ is OR₁₆ and R₁₆ is hydrogen(such that R₃ is hydroxy).

In some of any of the embodiments described herein, R₇ is alkyl and insome embodiments it is a lower alkyl, of 1-4 carbon atoms.

In some embodiments, R₇ is an alkyl such as ethyl, propyl, butyl,isopropyl, isobutyl, tert-butyl, each being optionally substituted.

In some embodiments, R₇ is methyl or ethyl, and is preferably asubstituted methyl or ethyl. In some of these embodiments, the methyl orethyl is substituted by, for example, a cycloalkyl or aryl. Suchsubstituents are also referred to in the art as alkylcycloalkyl andalkaryl, respectively. An exemplary alkaryl is benzyl (—CH₂-Phenyl).

In some embodiments, R₇ is propyl or isopropyl.

In some embodiments, R₇ is benzyl.

In some of any of the embodiments described herein, R₇ is acell-permealizable group, as defined herein, and in some embodiments, R₇is guanidyl.

In some of any of the embodiments described herein, R₁ is alkyl,cycloalkyl or aryl, and is preferably alkyl, as defined herein, and R₇is alkyl, as defined herein, preferably, ethyl, propyl, isopropyl orbenzyl.

In some of any of the embodiments described herein, R₁ is alkyl,cycloalkyl or aryl, and is preferably alkyl, as defined herein; R₇ isalkyl, as defined herein, preferably, ethyl, propyl, isopropyl orbenzyl; and R₃ is hydrogen.

In some of any of the embodiments described herein, R₁ is alkyl,cycloalkyl or aryl, and is preferably alkyl, as defined herein; R₇ is acell-permealizing group, as defined herein, preferably, guanidine orguanine; and R₃ is hydrogen.

In some of any of the embodiments described herein, R₁ is alkyl,cycloalkyl or aryl, and is preferably alkyl, as defined herein; R₇ is acell-permealizing group, as defined herein, preferably, guanidine orguanine, more preferably guanidine (guanidinyl).

Exemplary pseudo-disaccharide compounds are Compounds NB144, NB145,NB146 and NB150 (see, Table 1).

In some of any of the embodiments described herein, R₇ is hydrogen or amoiety such as (S)-4-amino-2-hydroxybutyryl (AHB), or(S)-4-amino-2-hydroxypropionyl (AHP).

In some of these embodiments, a modified amine is introduced to thecompound within a third saccharide moiety (Ring III; e.g., as R₅ inFormula Ia).

Any of the embodiments described herein for Formula Ia, and anycombination thereof, are included within the embodiments relating toFormula Ib.

In some of any of the embodiments of Formula Ib, R₁ is alkyl, as definedherein.

In some of any of the embodiments of Formula Ib, R₂ and R₇ are asdescribed in any of the respective embodiments for Formula Ia.

In some of any of the embodiments of Formula Ib, R₃, R₄ and R₆ are eachhydrogen.

In some of these embodiments, R₇ is alkyl, cycloalkyl or aryl, and ispreferably alkyl, as described herein.

In some embodiments, R₁ is alkyl, cycloalkyl or aryl, and is preferablyalkyl, as defined herein; R₇ is alkyl, as defined herein, preferably,ethyl, propyl, isopropyl or benzyl; and R₅ is a monosaccharide moiety ofFormula II, wherein R₁₄ and R₁₅ are both hydrogen.

An exemplary compound is NB147 (see, Table 1).

In some of any of the embodiments of Formula Ib, R₇ is hydrogen, acyl oramino-substituted α-hydroxy-acyl, as defined herein.

In some of these embodiments, X is NR₁₄R₁₅; and one of R₁₄ and R₁₅ isother than hydrogen. In some of these embodiments, one of R₁₄ and R₁₅ isa cell-permealizable group such as, for example, a guanidine group.Alternatively, one of R₁₄ and R₁₅ is alkyl, cycloalkyl or aryl, asdefined, for example, for any of the embodiments of R₇.

In some of any of the embodiments described herein, R₁ is alkyl,cycloalkyl or aryl, and is preferably alkyl, as defined herein; R₇ ishydrogen or amino-substituted α-hydroxy-acyl, as defined herein; R₅ is amonosaccharide moiety of Formula II; X is NR₁₄R₁₅; and R₁₅ is aguanidine group (guanidinyl; guanidyl).

In some of these embodiments, R₁₄ is hydrogen.

Exemplary compounds are NB151 and NB152 (see, Table 1).

In some of any of the embodiments described herein for Formula Ib, X isNR₁₄R15; and R₁₄ is hydrogen or methyl, unless specifically indicatedotherwise.

In some of any of the embodiments described herein for Formula Ib, X isNR₁₄R15; and R₁₄ is hydrogen.

In some of any of the embodiments described herein for Formula Ib, X isNR₁₄R15; and R₁₅ is acyl, as defined herein.

In some of any of the embodiments described herein for Formula Ib, X isNR₁₄R15; and one or both of R₁₄ and R₁₅ is a substituted orunsubstituted alkyl, a substituted or unsubstituted cycloalkyl, asubstituted or unsubstituted aryl, a substituted or unsubstitutedalkaryl, or a substituted or unsubstituted heteroaryl, or an acyl, asthese terms are defined herein.

In some of any of the embodiments described herein for Formula Ib, X isNR₁₄R15; and R₁₄ and R₁₅ form together a nitrogen-containingheterocyclic ring, such as, but not limited to, morpholine, piperidine,and piperazine.

In some of any of the embodiments described herein for Formula Ib, X isOR₁₃; and R₁₃ is as defined herein for R₁₆, in any of the respectiveembodiments. In some of these embodiments, R₁₃ is an acyl, forming anester at the 5″ position, as described herein in any of the respectiveembodiments.

In some of any one of the embodiments described herein for Formulae Iaand Ib, and any combination thereof, the stereoconfiguration at position6′ is an R configuration.

In some of any one of the embodiments described herein for Formula Ib,and any combination thereof, the stereoconfiguration at position 5″ isan S configuration.

Table 1 below presents exemplary compounds according to some embodimentsof the present invention.

TABLE 1 Compound Structure NB144

NB145

NB146

NB147

NB150

NB151

NB152

Additional exemplary compounds include compounds referred to herein asNB153, NB155, NB156, NB157, NB154, NB158, and NB159, the structures ofwhich are presented in the Examples section that follows.

Additional exemplary compounds include compounds referred to herein asmulti-esterified compounds, in which one or more, or two or more, ofR₃-R₆ is OR₁₆, and one or more of the R₁₆, R₂ and R₁₃, if present, isindependently an acyl, forming an ester at the respective position, suchthat the compound comprises at least two esters. The structures ofexemplary such are presented in Scheme 14 in the Examples section thatfollows.

According to some of any of the embodiments of the present invention,excluded from the scope of the present invention are compounds known inthe art, including any of the documents cited in the Background sectionof the instant application, which are encompassed by Formula Ia or Ib.

Exemplary compounds which are excluded from the scope of the presentembodiments include, but are not limited to, gentamicin, geneticin,fortimycin, apramycin, arbekacin, dibekacin, geneticin (G-418, G418),habekacin, kanamycin, Lividomycin, paromomycin, streptomycin andtobramycin.

Additional exemplary compounds which are excluded from the scope of thepresent embodiments include compounds represented by Formula Ia, inwhich R₂ is hydrogen, and R₇ is hydrogen, AHB or AHP, or equivalents ofAHB and AHP, as defined in WO 2007/113841 and WO 2012/066546; andcompounds represented by Formula Ib, in which R₂ is hydrogen, R₇ ishydrogen, AHB or AHP, or equivalents of AHB and AHP, as defined in WO2007/113841 and WO 2012/066546, and R₁₄ and R₁₅ are each hydrogen.

According to some embodiments of the present invention, when R₂ ishydrogen, then R₇ is not hydrogen, AHB or AHP, or equivalents of AHB andAHP, as defined in WO 2007/113841 and WO 2012/066546, and/or one or bothof R₁₄ and R₁₅, if present, is not hydrogen.

According to some embodiments of the present invention, one or both ofthe amine substituents at positions 1 or 5″ of the aminoglycosidestructure is a modified amine, as defined herein, such that R₇ and/orone or both of R₁₄ and R₁₅ is not hydrogen.

The chemical structures of exemplary compounds which are excluded fromthe scope of the present invention are presented in Table 2 below.

TABLE 2 Number Structure n/a

NB30

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

NB54

NB74

NB84

NB118

NB119

NB122

NB123

NB124

NB125

NB127

NB128

According to some embodiments of the present invention, excluded fromthe scope of the present embodiments are also compounds represented byFormulae I′ a as follows:

wherein:

the dashed line indicates a stereo-configuration of position 6′ being anR configuration or an S configuration;

R′₁ is alkyl, cycloalkyl, alkaryl or aryl;

R′₂ is OR′, wherein R′ is selected from hydrogen, a substituted orunsubstituted alkyl, a substituted or unsubstituted cycloalkyl, asubstituted or unsubstituted aryl, a substituted or unsubstitutedheteroaryl, a substituted or unsubstituted alkaryl, and an acyl, asdefined herein;

R′₄ is selected from hydrogen, acyl, an amino-substituted alpha-hydroxyacyl, a substituted or unsubstituted alkyl, a substituted orunsubstituted cycloalkyl, a substituted or unsubstituted aryl, asubstituted or unsubstituted alkaryl and a cell-permealizable group,such as guanyl or guanidyl; and

R′₃ is hydrogen or a monosaccharide moiety represented by Formula II′:

wherein the curved line denotes a position of attachment; and

R′₅ and R′₆ are each independently selected from hydrogen, a substitutedor unsubstituted alkyl, a substituted or unsubstituted cycloalkyl, asubstituted or unsubstituted aryl, a substituted or unsubstitutedalkaryl, a substituted or unsubstituted heteroaryl, acyl, and acell-permealizable group such as guanyl and guanidinyl, or,alternatively, R′₅ and R′₆ form together a heterocyclic ring,

wherein when R′₂ is hydrogen, R′₄ is not hydrogen, AHB or AHP, and/or atleast one of R′₅ and/or R′₆, if present, is not hydrogen.

According to some embodiments of the present invention, excluded fromthe scope of the present embodiments are also compounds represented byFormulae I′b as follows:

or a pharmaceutically acceptable salt thereof,

wherein:

the dashed line indicates a stereo-configuration of position 6′ being anR configuration or an S configuration;

R′₁ is selected from hydrogen, alkyl, cycloalkyl or aryl;

R₂ is OR′, wherein R′ is selected from hydrogen, a substituted orunsubstituted alkyl, a substituted or unsubstituted cycloalkyl, asubstituted or unsubstituted aryl, a substituted or unsubstitutedheteroaryl, a substituted or unsubstituted alkaryl, and an acyl;

R′₄ is selected from hydrogen, acyl, an amino-substituted alpha-hydroxyacyl, a substituted or unsubstituted alkyl, a substituted orunsubstituted cycloalkyl, a substituted or unsubstituted aryl, asubstituted or unsubstituted alkaryl, and a cell-permealizable group;

R′₆ and R′₇ are each independently selected from hydrogen, a substitutedor unsubstituted alkyl, a substituted or unsubstituted cycloalkyl, asubstituted or unsubstituted aryl, a substituted or unsubstitutedalkaryl, a substituted or unsubstituted heteroaryl, acyl, and acell-permealizable group, or, alternatively, R′₅ and R′₆ form together aheterocyclic ring; and

R′₈ is alkyl, cycloalkyl or aryl,

wherein when R′₂ is hydrogen, R′₄ is not hydrogen, AHB or AHP, and/or atleast one of R′₆ and/or R′₇, if present, is not hydrogen.

In some of any of the embodiments described herein, excluded from thescope of the present invention are the compounds presented in Table 1.

According to some of any of the embodiments described herein, a compoundas described herein is represented by Formula Ic:

or a pharmaceutically acceptable salt thereof,

wherein:

the dashed line indicates a stereo-configuration of position 6′ being anR configuration or an S configuration;

X₁ is O or S;

Rx, Ry1 and Rz are each independently hydrogen, alkyl or cycloalkyl;

Ry2-Ry9 and Rw1-Rw3 are each independently selected from hydrogen,alkyl, alkenyl, alkynyl, aryl, heteroaryl and cycloalkyl, each beingsubstituted or unsubstituted, or, alternatively, each can be as definedherein for R₇-R₉;

R₁ is a substituted or unsubstituted hydroxy alkyl (e.g., —CH₂—OH);

R₂ is selected from the group consisting of hydrogen, a substituted orunsubstituted alkyl, a substituted or unsubstituted alkenyl, asubstituted or unsubstituted alkynyl, a substituted or unsubstitutedcycloalkyl, a substituted or unsubstituted aryl, a substituted orunsubstituted heteroaryl, a substituted or unsubstituted alkaryl andacyl, as described herein for Formula Ia;

R₃-R₆ are each independently selected from the group consisting ofhydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heteroalicyclic, aryl,heteroaryl, amine and OR₁₆, wherein R₁₆ is independently selected fromhydrogen, a monosaccharide moiety, an oligosaccharide moiety, asubstituted or unsubstituted alkyl, a substituted or unsubstitutedalkenyl, a substituted or unsubstituted alkynyl, a substituted orunsubstituted cycloalkyl, a substituted or unsubstituted aryl, asubstituted or unsubstituted heteroaryl, a substituted or unsubstitutedalkaryl and acyl, as described herein for any of the respectiveembodiments of Formula Ia; and

R₇-R₉ are each independently selected from the group consisting ofhydrogen, acyl, an amino-substituted alpha-hydroxy acyl, a substitutedor unsubstituted alkyl, a substituted or unsubstituted alkenyl, asubstituted or unsubstituted alkynyl, a substituted or unsubstitutedcycloalkyl, a substituted or unsubstituted aryl, a substituted orunsubstituted alkaryl, carboxylate, sulfonyl (including alkyl sulfonyland aryl sulfonyl) and a cell-permealizable group, as described hereinfor any of the respective embodiments of Formula Ia.

In some of these embodiments, one or more R₃-R₆ is a monosaccharide oran oligosaccharide, as described herein for any of the respectiveembodiments of Formulae Ia and Ib.

In some of any of the embodiments of Formula Ic:

X₁ is O;

Rx, Ry1 and Rz are each hydrogen;

Ry2-Ry9 and Rw1-Rw3 are each hydrogen;

R₂ is selected from the group consisting of hydrogen, a substituted orunsubstituted alkyl, a substituted or unsubstituted alkenyl, asubstituted or unsubstituted alkynyl, a substituted or unsubstitutedcycloalkyl, a substituted or unsubstituted aryl, a substituted orunsubstituted heteroaryl, a substituted or unsubstituted alkaryl andacyl, as described herein for Formula Ia or Ib;

R₃-R₆ are each independently OR₁₆, wherein R₁₆ is independently selectedfrom hydrogen, a monosaccharide moiety, an oligosaccharide moiety, asubstituted or unsubstituted alkyl, a substituted or unsubstitutedalkenyl, a substituted or unsubstituted alkynyl, a substituted orunsubstituted cycloalkyl, a substituted or unsubstituted aryl, asubstituted or unsubstituted heteroaryl, a substituted or unsubstitutedalkaryl and acyl, as described herein for any of the respectiveembodiments of Formula Ia; and

R₇-R₉ are each independently selected from the group consisting ofhydrogen, acyl, an amino-substituted alpha-hydroxy acyl, a substitutedor unsubstituted alkyl, a substituted or unsubstituted alkenyl, asubstituted or unsubstituted alkynyl, a substituted or unsubstitutedcycloalkyl, a substituted or unsubstituted aryl, a substituted orunsubstituted alkaryl, carboxylate, sulfonyl (including alkyl sulfonyland aryl sulfonyl) and a cell-permealizable group, as described hereinfor any of the respective embodiments of Formula Ia or Ib.

In some of any of the embodiments of Formula Ic:

X₁ is O;

Rx, Ry1 and Rz are each hydrogen;

Ry2-Ry9 and Rw1-Rw3 are each hydrogen;

R₂ is hydrogen;

R₃-R₆ are each independently OR₁₆, wherein R₁₆ is hydrogen; and

R₇-R₉ are each independently selected from the group consisting ofhydrogen, acyl, an amino-substituted alpha-hydroxy acyl, a substitutedor unsubstituted alkyl, a substituted or unsubstituted alkenyl, asubstituted or unsubstituted alkynyl, a substituted or unsubstitutedcycloalkyl, a substituted or unsubstituted aryl, a substituted orunsubstituted alkaryl, and a cell-permealizable group, as describedherein for any of the respective embodiments of Formula Ia or Ib.

In some of any of the embodiments of Formula Ic:

X₁ is O;

Rx, Ry1 and Rz are each hydrogen;

Ry2-Ry9 and Rw1-Rw3 are each hydrogen;

R₂ is hydrogen;

R₃, R₄ and R₆ are each independently OR₁₆, wherein R₁₆ is hydrogen;

R₇ is a monosaccharide represented by Formula II as described herein,wherein X₂ is preferably NR₁₄R₁₅, as described herein for any of therespective embodiments of Formula Ia or Ib; and

R₇-R₉ are each independently selected from the group consisting ofhydrogen, acyl, an amino-substituted alpha-hydroxy acyl, a substitutedor unsubstituted alkyl, a substituted or unsubstituted alkenyl, asubstituted or unsubstituted alkynyl, a substituted or unsubstitutedcycloalkyl, a substituted or unsubstituted aryl, a substituted orunsubstituted alkaryl, and a cell-permealizable group, as describedherein for any of the respective embodiments of Formula Ia or Ib.

Compounds represented by Formula Ic are also referred to herein as“diol-containing” compounds.

Exemplary compounds encompassed by Formula Ic include NB153, NB155,NB156 and NB157, the structures of which are presented in the Examplessection that follows.

According to some of any of the embodiments described herein, a compoundas described herein is represented by Formula Id:

or a pharmaceutically acceptable salt thereof,

wherein:

the dashed line indicates a stereo-configuration of position 6′ being anR configuration or an S configuration;

X₁ is O or S;

Rx, Ry1-Ry9 and Rw1-Rw3 are each independently selected from hydrogen,alkyl, alkenyl, alkynyl, aryl, heteroaryl and cycloalkyl, each beingsubstituted or unsubstituted, or, alternatively, each can be as definedherein for R₇-R₉;

R₁ is selected from the group consisting of hydrogen, a substituted orunsubstituted alkyl, a substituted or unsubstituted alkenyl, asubstituted or unsubstituted alkynyl, a substituted or unsubstitutedcycloalkyl, a substituted or unsubstituted aryl, a substituted orunsubstituted heteroaryl, a substituted or unsubstituted alkaryl, asubstituted or unsubstituted amine, a substituted or unsubstitutedamide, an acyl, a carboxylate, and a saturated or unsaturated and/orsubstituted or unsubstituted hydroxy alkyl (e.g., —CH₂—OH), as describedherein in any of the respective embodiments of Formula Ia or Ib;

R₂ is selected from the group consisting of hydrogen, a substituted orunsubstituted alkyl, a substituted or unsubstituted alkenyl, asubstituted or unsubstituted alkynyl, a substituted or unsubstitutedcycloalkyl, a substituted or unsubstituted aryl, a substituted orunsubstituted heteroaryl, a substituted or unsubstituted alkaryl andacyl, as described herein in any of the respective embodiments ofFormula Ia or Ib;

R₄-R₆ are each independently selected from the group consisting ofhydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heteroalicyclic, aryl,heteroaryl, amine and OR₁₆, wherein R₁₆ is independently selected fromhydrogen, a monosaccharide moiety, an oligosaccharide moiety, asubstituted or unsubstituted alkyl, a substituted or unsubstitutedalkenyl, a substituted or unsubstituted alkynyl, a substituted orunsubstituted cycloalkyl, a substituted or unsubstituted aryl, asubstituted or unsubstituted heteroaryl, a substituted or unsubstitutedalkaryl and acyl, as described herein in any of the respectiveembodiments of Formula Ia or Ib; and

R₇-R₉ are each independently selected from the group consisting ofhydrogen, acyl, an amino-substituted alpha-hydroxy acyl, a substitutedor unsubstituted alkyl, a substituted or unsubstituted alkenyl, asubstituted or unsubstituted alkynyl, a substituted or unsubstitutedcycloalkyl, a substituted or unsubstituted aryl, a substituted orunsubstituted alkaryl, carboxylate, sulfonyl (including alkyl sulfonyland aryl sulfonyl) and a cell-permealizable group as defined herein, asdescribed herein in any of the respective embodiments of Formula Ia orIb.

In some of these embodiments, one or more R₄-R₆ is a monosaccharide oran oligosaccharide, as described herein for any of the respectiveembodiments of Formulae Ia and Ib.

In some of any of the embodiments of Formula Id:

X₁ is O;

Rx, Ry1-Ry9 and Rw1-Rw3 are each hydrogen;

R₁ is selected from the group consisting of hydrogen, a substituted orunsubstituted alkyl, a substituted or unsubstituted alkenyl, asubstituted or unsubstituted alkynyl, a substituted or unsubstitutedcycloalkyl, a substituted or unsubstituted aryl, a substituted orunsubstituted heteroaryl, a substituted or unsubstituted alkaryl, asubstituted or unsubstituted amine, a substituted or unsubstitutedamide, an acyl, a carboxylate, and a saturated or unsaturated and/orsubstituted or unsubstituted hydroxy alkyl (e.g., —CH₂—OH), as describedherein in any of the respective embodiments of Formula Ia or Ib;

R₂ is selected from the group consisting of hydrogen, a substituted orunsubstituted alkyl, a substituted or unsubstituted alkenyl, asubstituted or unsubstituted alkynyl, a substituted or unsubstitutedcycloalkyl, a substituted or unsubstituted aryl, a substituted orunsubstituted heteroaryl, a substituted or unsubstituted alkaryl andacyl, as described herein for Formula Ia or Ib;

R₄-R₆ are each independently OR₁₆, wherein R₁₆ is independently selectedfrom hydrogen, a monosaccharide moiety, an oligosaccharide moiety, asubstituted or unsubstituted alkyl, a substituted or unsubstitutedalkenyl, a substituted or unsubstituted alkynyl, a substituted orunsubstituted cycloalkyl, a substituted or unsubstituted aryl, asubstituted or unsubstituted heteroaryl, a substituted or unsubstitutedalkaryl and acyl, as described herein for any of the respectiveembodiments of Formula Ia; and

R₇-R₉ are each independently selected from the group consisting ofhydrogen, acyl, an amino-substituted alpha-hydroxy acyl, a substitutedor unsubstituted alkyl, a substituted or unsubstituted alkenyl, asubstituted or unsubstituted alkynyl, a substituted or unsubstitutedcycloalkyl, a substituted or unsubstituted aryl, a substituted orunsubstituted alkaryl, carboxylate, sulfonyl (including alkyl sulfonyland aryl sulfonyl) and a cell-permealizable group, as described hereinfor any of the respective embodiments of Formula Ia or Ib.

In some of any of the embodiments of Formula Id:

X₁ is O;

Rx, Ry1-Ry9 and Rw1-Rw3 are each hydrogen;

R₁ is hydrogen, alkyl, cycloalkyl or aryl, as described herein, and ispreferably hydrogen or a lower alkyl, as described herein;

R₂ is hydrogen;

R₄-R₆ are each independently OR₁₆, wherein R₁₆ is hydrogen; and

R₇-R₉ are each independently selected from the group consisting ofhydrogen, acyl, an amino-substituted alpha-hydroxy acyl, a substitutedor unsubstituted alkyl, a substituted or unsubstituted alkenyl, asubstituted or unsubstituted alkynyl, a substituted or unsubstitutedcycloalkyl, a substituted or unsubstituted aryl, a substituted orunsubstituted alkaryl, and a cell-permealizable group, as describedherein for any of the respective embodiments of Formula Ia or Ib.

In some of any of the embodiments of Formula Id:

X₁ is O;

Rx, Ry1-Ry9 and Rw1-Rw3 are each hydrogen;

R₁ is hydrogen, alkyl, cycloalkyl or aryl, as described herein, and ispreferably hydrogen or a lower alkyl, as described herein;

R₂ is hydrogen;

R₄ and R₆ are each independently OR₁₆, wherein R₁₆ is hydrogen;

R₅ is a monosaccharide represented by Formula II as described herein,wherein X₂ is preferably NR₁₄R₁₅, as described herein for any of therespective embodiments of Formula Ia or Ib; and

R₇-R₉ are each independently selected from the group consisting ofhydrogen, acyl, an amino-substituted alpha-hydroxy acyl, a substitutedor unsubstituted alkyl, a substituted or unsubstituted alkenyl, asubstituted or unsubstituted alkynyl, a substituted or unsubstitutedcycloalkyl, a substituted or unsubstituted aryl, a substituted orunsubstituted alkaryl, and a cell-permealizable group, as describedherein for any of the respective embodiments of Formula Ia or Ib.

Compounds represented by Formula Id are also referred to herein as“unsaturated Glucosamine (Ring I)-containing” compound. Exemplarycompounds encompassed by Formula Id include NB154, NB158 and NB159, thestructures of which are presented in the Examples section that follows.

According to some of any of the embodiments described herein, a compoundas described herein is represented by Formula Ie:

or a pharmaceutically acceptable salt thereof,

wherein:

the dashed line indicates a stereo-configuration of position 6′ being anR configuration or an S configuration;

X₁ is O or S;

Rx, Ry1 and Rz are each independently hydrogen, alkyl or cycloalkyl;

Ry2-Ry9 and Rw1-Rw3 are each independently selected from hydrogen,alkyl, alkenyl, alkynyl, aryl, heteroaryl and cycloalkyl, each beingsubstituted or unsubstituted, or, alternatively, each can be as definedherein for R₇-R₉;

R₁ is selected from the group consisting of hydrogen, a substituted orunsubstituted alkyl, a substituted or unsubstituted alkenyl, asubstituted or unsubstituted alkynyl, a substituted or unsubstitutedcycloalkyl, a substituted or unsubstituted aryl, a substituted orunsubstituted heteroaryl, a substituted or unsubstituted alkaryl, asubstituted or unsubstituted amine, a substituted or unsubstitutedamide, an acyl, a carboxylate, and a saturated or unsaturated and/orsubstituted or unsubstituted hydroxy alkyl (e.g., —CH₂—OH), as describedherein in any of the respective embodiments of Formula Ia or Ib;

R₂ is selected from the group consisting of hydrogen, a substituted orunsubstituted alkyl, a substituted or unsubstituted alkenyl, asubstituted or unsubstituted alkynyl, a substituted or unsubstitutedcycloalkyl, a substituted or unsubstituted aryl, a substituted orunsubstituted heteroaryl, a substituted or unsubstituted alkaryl andacyl, as described herein for Formula Ia;

R₃-R₆ are each independently selected from the group consisting ofhydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heteroalicyclic, aryl,heteroaryl, amine and OR₁₆, wherein R₁₆ is independently selected fromhydrogen, a monosaccharide moiety, an oligosaccharide moiety, asubstituted or unsubstituted alkyl, a substituted or unsubstitutedalkenyl, a substituted or unsubstituted alkynyl, a substituted orunsubstituted cycloalkyl, a substituted or unsubstituted aryl, asubstituted or unsubstituted heteroaryl, a substituted or unsubstitutedalkaryl and acyl, as described herein for any of the respectiveembodiments of Formula Ia, wherein at least one of R₃-R₆ is OR₁₆; and

R₇-R₉ are each independently selected from the group consisting ofhydrogen, acyl, an amino-substituted alpha-hydroxy acyl, a substitutedor unsubstituted alkyl, a substituted or unsubstituted alkenyl, asubstituted or unsubstituted alkynyl, a substituted or unsubstitutedcycloalkyl, a substituted or unsubstituted aryl, a substituted orunsubstituted alkaryl, carboxylate, sulfonyl (including alkyl sulfonyland aryl sulfonyl) and a cell-permealizable group, as described hereinfor any of the respective embodiments of Formula Ia or Ib,

wherein at least two of R₂ and OR₁₆ in the one or more of R₃-R₆ whichis/are OR₁₆ is an acyl.

In some of these embodiments, one or more R₃-R₆ is a monosaccharide oran oligosaccharide, as described herein for any of the respectiveembodiments of Formulae Ia and Ib.

In some of any of the embodiments of Formula Ie:

X₁ is O;

Rx, Ry1 and Rz are each hydrogen;

Ry2-Ry9 and Rw1-Rw3 are each hydrogen;

R₁ is selected from the group consisting of hydrogen, a substituted orunsubstituted alkyl, a substituted or unsubstituted alkenyl, asubstituted or unsubstituted alkynyl, a substituted or unsubstitutedcycloalkyl, a substituted or unsubstituted aryl, a substituted orunsubstituted heteroaryl, a substituted or unsubstituted alkaryl, asubstituted or unsubstituted amine, a substituted or unsubstitutedamide, an acyl, a carboxylate, and a saturated or unsaturated and/orsubstituted or unsubstituted hydroxy alkyl (e.g., —CH₂—OH), as describedherein in any of the respective embodiments of Formula Ia or Ib;

R₂ is an acyl;

R₃-R₆ are each independently OR₁₆, wherein R₁₆ is independently selectedfrom hydrogen, a monosaccharide moiety, an oligosaccharide moiety, asubstituted or unsubstituted alkyl, a substituted or unsubstitutedalkenyl, a substituted or unsubstituted alkynyl, a substituted orunsubstituted cycloalkyl, a substituted or unsubstituted aryl, asubstituted or unsubstituted heteroaryl, a substituted or unsubstitutedalkaryl and acyl, as described herein for any of the respectiveembodiments of Formula Ia, at least one of R₃-R₆ is OR₁₆ in which R₁₆ isan acyl; and

R₇-R₉ are each independently selected from the group consisting ofhydrogen, acyl, an amino-substituted alpha-hydroxy acyl, a substitutedor unsubstituted alkyl, a substituted or unsubstituted alkenyl, asubstituted or unsubstituted alkynyl, a substituted or unsubstitutedcycloalkyl, a substituted or unsubstituted aryl, a substituted orunsubstituted alkaryl, carboxylate, sulfonyl (including alkyl sulfonyland aryl sulfonyl) and a cell-permealizable group, as described hereinfor any of the respective embodiments of Formula Ia or Ib.

In some of any of the embodiments of Formula Ie:

X₁ is O;

Rx, Ry1 and Rz are each hydrogen;

Ry2-Ry9 and Rw1-Rw3 are each hydrogen;

R₁ is hydrogen, alkyl, cycloalkyl or aryl, and is preferably hydrogen ora lower alkyl, as described herein;

R₂ is acyl;

R₃, R₅ and R₆ are each independently OR₁₆, wherein R₁₆ is acyl;

R₄ is OR₁₆, wherein R₁₆ is hydrogen or acyl; and

R₇-R₉ are each independently selected from the group consisting ofhydrogen, acyl, an amino-substituted alpha-hydroxy acyl, a substitutedor unsubstituted alkyl, a substituted or unsubstituted alkenyl, asubstituted or unsubstituted alkynyl, a substituted or unsubstitutedcycloalkyl, a substituted or unsubstituted aryl, a substituted orunsubstituted alkaryl, and a cell-permealizable group, as describedherein for any of the respective embodiments of Formula Ia or Ib.

In some of any of the embodiments of Formula Ie:

X₁ is O;

Rx, Ry1 and Rz are each hydrogen;

Ry2-Ry9 and Rw1-Rw3 are each hydrogen;

R₁ is hydrogen, alkyl, cycloalkyl or aryl, and is preferably hydrogen ora lower alkyl, as described herein;

R₂ is acyl;

R₃ and R₆ are each independently OR₁₆, wherein R₁₆ is an acyl;

R₄ is OR₁₆, wherein R₁₆ is hydrogen or acyl; and

R₅ is a monosaccharide represented by Formula II as described herein,wherein X₂ is preferably NR₁₄R₁₅, as described herein for any of therespective embodiments of Formula Ia or Ib; and

R₇-R₉ are each independently selected from the group consisting ofhydrogen, acyl, an amino-substituted alpha-hydroxy acyl, a substitutedor unsubstituted alkyl, a substituted or unsubstituted alkenyl, asubstituted or unsubstituted alkynyl, a substituted or unsubstitutedcycloalkyl, a substituted or unsubstituted aryl, a substituted orunsubstituted alkaryl, and a cell-permealizable group, as describedherein for any of the respective embodiments of Formula Ia or Ib.

Compounds represented by Formula Ie are also referred to herein as“multi-esterified” compounds.

Exemplary compounds encompassed by Formula Ie include the compoundspresented in Scheme 14 in the Examples section that follows.

For any of the embodiments described herein, and any combinationthereof, the compound may be in a form of a salt, for example, apharmaceutically acceptable salt.

As used herein, the phrase “pharmaceutically acceptable salt” refers toa charged species of the parent compound and its counter-ion, which istypically used to modify the solubility characteristics of the parentcompound and/or to reduce any significant irritation to an organism bythe parent compound, while not abrogating the biological activity andproperties of the administered compound. A pharmaceutically acceptablesalt of a compound as described herein can alternatively be formedduring the synthesis of the compound, e.g., in the course of isolatingthe compound from a reaction mixture or re-crystallizing the compound.

In the context of some of the present embodiments, a pharmaceuticallyacceptable salt of the compounds described herein may optionally be anacid addition salt comprising at least one basic (e.g., amine and/orguanidine) group of the compound which is in a positively charged form(e.g., wherein the basic group is protonated), in combination with atleast one counter-ion, derived from the selected base, that forms apharmaceutically acceptable salt.

The acid addition salts of the compounds described herein may thereforebe complexes formed between one or more basic groups of the compound andone or more equivalents of an acid.

Depending on the stoichiometric proportions between the charged group(s)in the compound and the counter-ion in the salt, the acid additionssalts can be either mono-addition salts or poly-addition salts.

The phrase “mono-addition salt”, as used herein, refers to a salt inwhich the stoichiometric ratio between the counter-ion and charged formof the compound is 1:1, such that the addition salt includes one molarequivalent of the counter-ion per one molar equivalent of the compound.

The phrase “poly-addition salt”, as used herein, refers to a salt inwhich the stoichiometric ratio between the counter-ion and the chargedform of the compound is greater than 1:1 and is, for example, 2:1, 3:1,4:1 and so on, such that the addition salt includes two or more molarequivalents of the counter-ion per one molar equivalent of the compound.

An example, without limitation, of a pharmaceutically acceptable saltwould be an ammonium cation or guanidinium cation and an acid additionsalt thereof.

The acid addition salts may include a variety of organic and inorganicacids, such as, but not limited to, hydrochloric acid which affords ahydrochloric acid addition salt, hydrobromic acid which affords ahydrobromic acid addition salt, acetic acid which affords an acetic acidaddition salt, ascorbic acid which affords an ascorbic acid additionsalt, benzenesulfonic acid which affords a besylate addition salt,camphorsulfonic acid which affords a camphorsulfonic acid addition salt,citric acid which affords a citric acid addition salt, maleic acid whichaffords a maleic acid addition salt, malic acid which affords a malicacid addition salt, methanesulfonic acid which affords a methanesulfonicacid (mesylate) addition salt, naphthalenesulfonic acid which affords anaphthalenesulfonic acid addition salt, oxalic acid which affords anoxalic acid addition salt, phosphoric acid which affords a phosphoricacid addition salt, toluenesulfonic acid which affords ap-toluenesulfonic acid addition salt, succinic acid which affords asuccinic acid addition salt, sulfuric acid which affords a sulfuric acidaddition salt, tartaric acid which affords a tartaric acid addition saltand trifluoroacetic acid which affords a trifluoroacetic acid additionsalt. Each of these acid addition salts can be either a mono-additionsalt or a poly-addition salt, as these terms are defined herein.

The present embodiments further encompass any enantiomers,diastereomers, prodrugs, solvates, hydrates and/or pharmaceuticallyacceptable salts of the compounds described herein.

As used herein, the term “enantiomer” refers to a stereoisomer of acompound that is superposable with respect to its counterpart only by acomplete inversion/reflection (mirror image) of each other. Enantiomersare said to have “handedness” since they refer to each other like theright and left hand. Enantiomers have identical chemical and physicalproperties except when present in an environment which by itself hashandedness, such as all living systems. In the context of the presentembodiments, a compound may exhibit one or more chiral centers, each ofwhich exhibiting an R- or an S-configuration and any combination, andcompounds according to some embodiments of the present invention, canhave any their chiral centers exhibit an R- or an S-configuration.

The term “diastereomers”, as used herein, refers to stereoisomers thatare not enantiomers to one another. Diastereomerism occurs when two ormore stereoisomers of a compound have different configurations at one ormore, but not all of the equivalent (related) stereocenters and are notmirror images of each other. When two diastereoisomers differ from eachother at only one stereocenter they are epimers. Each stereo-center(chiral center) gives rise to two different configurations and thus totwo different stereoisomers. In the context of the present invention,embodiments of the present invention encompass compounds with multiplechiral centers that occur in any combination of stereo-configuration,namely any diastereomer.

According to some of any of the embodiments described herein, astereo-configuration of each of position 6′ and position 5″ (if present)is independently an R configuration or an S configuration.

According to some of any of the embodiments described herein, astereo-configuration of position 6′ is an R configuration.

According to some of any of the embodiments described herein, astereo-configuration of position 5″, if present, is an S configuration.

According to some of any of the embodiments described herein, astereo-configuration of position 6′ is an R configuration and astereo-configuration of position 5″, if preset, is an R configuration oran S configuration.

According to some of any of the embodiments described herein, astereo-configuration of position 6′ is an R configuration and astereo-configuration of position 5″, if present, is an S configuration.

The term “prodrug” refers to an agent, which is converted into theactive compound (the active parent drug) in vivo. Prodrugs are typicallyuseful for facilitating the administration of the parent drug. They may,for instance, be bioavailable by oral administration whereas the parentdrug is not. A prodrug may also have improved solubility as comparedwith the parent drug in pharmaceutical compositions. Prodrugs are alsooften used to achieve a sustained release of the active compound invivo. An example, without limitation, of a prodrug would be a compoundof the present invention, having one or more carboxylic acid moieties,which is administered as an ester (the “prodrug”). Such a prodrug ishydrolyzed in vivo, to thereby provide the free compound (the parentdrug). The selected ester may affect both the solubility characteristicsand the hydrolysis rate of the prodrug.

The term “solvate” refers to a complex of variable stoichiometry (e.g.,di-, tri-, tetra-, penta-, hexa-, and so on), which is formed by asolute (the compound of the present invention) and a solvent, wherebythe solvent does not interfere with the biological activity of thesolute. Suitable solvents include, for example, ethanol, acetic acid andthe like.

The term “hydrate” refers to a solvate, as defined hereinabove, wherethe solvent is water.

The terms “hydroxyl” or “hydroxy”, as used herein, refer to an —OHgroup.

As used herein, the term “amine” describes a —NR′R″ group where each ofR′ and R″ is independently hydrogen, alkyl, alkenyl, alkynyl,cycloalkyl, heteroalicyclic, aryl, heteroaryl, alkaryl, alkheteroaryl,or acyl, as these terms are defined herein. Alternatively, one or bothof R′ and R″ can be, for example, hydroxy, alkoxy, hydroxyalkyl,trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl,heteroalicyclic, amine, halide, sulfonate, sulfoxide, phosphonate,hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano,nitro, azo, sulfonamide, carbonyl, C-carboxylate, O-carboxylate,N-thiocarbamate, O-thiocarbamate, urea, thiourea, N-carbamate,O-carbamate, C-amide, N-amide, guanyl, guanidine and hydrazine.

As used herein, the term “alkyl” describes an aliphatic hydrocarbonincluding straight chain and branched chain groups. The alkyl may have 1to 20 carbon atoms, or 1-10 carbon atoms, and may be branched orunbranched. According to some embodiments of the present invention, thealkyl is a low (or lower) alkyl, having 1-4 carbon atoms (namely,methyl, ethyl, propyl and butyl).

Whenever a numerical range; e.g., “1-10”, is stated herein, it impliesthat the group, in this case the alkyl group, may contain 1 carbon atom,2 carbon atoms, 3 carbon atoms, etc., up to and including 10 carbonatoms. In some embodiments, the alkyl is a lower alkyl, including 1-6 or1-4 carbon atoms.

An alkyl can be substituted or unsubstituted. When substituted, thesubstituent can be, for example, one or more of an alkyl (forming abranched alkyl), an alkenyl, an alkynyl, a cycloalkyl, an aryl, aheteroaryl, a heteroalicyclic, a halo, a trihaloalkyl, a hydroxy, analkoxy and a hydroxyalkyl as these terms are defined hereinbelow. Analkyl substituted by aryl is also referred to herein as “alkaryl”, anexample of which is benzyl.

Whenever “alkyl” is described, it can be replaced also by alkenyl oralkynyl. The term “alkyl” as used herein, also encompasses saturated orunsaturated hydrocarbon, hence this term further encompasses alkenyl andalkynyl.

The term “alkenyl” describes an unsaturated alkyl, as defined herein,having at least two carbon atoms and at least one carbon-carbon doublebond, e.g., allyl, vinyl, 3-butenyl, 2-butenyl, 2-hexenyl andi-propenyl. The alkenyl may be substituted or unsubstituted by one ormore substituents, as described hereinabove.

The term “alkynyl”, as defined herein, is an unsaturated alkyl having atleast two carbon atoms and at least one carbon-carbon triple bond. Thealkynyl may be substituted or unsubstituted by one or more substituents,as described hereinabove.

The term “cycloalkyl” refers to an all-carbon monocyclic or fused ring(i.e., rings which share an adjacent pair of carbon atoms), branched orunbranched group containing 3 or more carbon atoms where one or more ofthe rings does not have a completely conjugated pi-electron system, andmay further be substituted or unsubstituted. Exemplary cycloalkyl groupsinclude, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,or cyclododecyl. The cycloalkyl can be substituted or unsubstituted.When substituted, the substituent can be, for example, one or more of analkyl, an alkenyl, an alkynyl, a cycloalkyl, an aryl, a heteroaryl, aheteroalicyclic, a halo, a trihaloalkyl, a hydroxy, an alkoxy and ahydroxyalkyl as these terms are defined hereinbelow.

The term “aryl” describes an all-carbon monocyclic or fused-ringpolycyclic (i.e., rings which share adjacent pairs of carbon atoms)groups having a completely conjugated pi-electron system. The aryl groupmay be unsubstituted or substituted by one or more substituents. Whensubstituted, the substituent can be, for example, one or more of analkyl, an alkenyl, an alkynyl, a cycloalkyl, an aryl, a heteroaryl, aheteroalicyclic, a halo, a trihaloalkyl, a hydroxy, an alkoxy and ahydroxyalkyl as these terms are defined hereinbelow.

The term “heteroaryl” describes a monocyclic or fused ring (i.e., ringswhich share an adjacent pair of atoms) group having in the ring(s) oneor more atoms, such as, for example, nitrogen, oxygen and sulfur and, inaddition, having a completely conjugated pi-electron system. Examples,without limitation, of heteroaryl groups include pyrrole, furane,thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine,quinoline, isoquinoline and purine. Representative examples arethiadiazole, pyridine, pyrrole, oxazole, indole, purine and the like.The heteroaryl group may be unsubstituted or substituted by one or moresubstituents. When substituted, the substituent can be, for example, oneor more of an alkyl, an alkenyl, an alkynyl, a cycloalkyl, an aryl, aheteroaryl, a heteroalicyclic, a halo, a trihaloalkyl, a hydroxy, analkoxy and a hydroxyalkyl as these terms are defined hereinbelow.

The term “heteroalicyclic”, as used herein, describes a monocyclic orfused ring group having in the ring(s) one or more atoms such asnitrogen, oxygen and sulfur. The rings may also have one or more doublebonds. However, the rings do not have a completely conjugatedpi-electron system. Representative examples are morpholine, piperidine,piperazine, tetrahydrofurane, tetrahydropyrane and the like. Theheteroalicyclic may be substituted or unsubstituted. When substituted,the substituent can be, for example, one or more of an alkyl, analkenyl, an alkynyl, a cycloalkyl, an aryl, a heteroaryl, aheteroalicyclic, a halo, a trihaloalkyl, a hydroxy, an alkoxy and ahydroxyalkyl as these terms are defined hereinbelow.

The term “halide”, as used herein, refers to the anion of a halo atom,i.e. F⁻, Cl⁻, Br⁻ and I⁻.

The term “halo” refers to F, Cl, Br and I atoms as substituents.

The term “alkoxide” refers to an R′—O⁻ anion, wherein R′ is as definedhereinabove.

The term “alkoxy” refers to an —OR′ group, wherein R′ is alkyl orcycloalkyl, as defined herein.

The term “aryloxy” refers to an —OR′ group, wherein R′ is aryl, asdefined herein.

The term “heteroaryloxy” refers to an —OR′ group, wherein R′ isheteroaryl, as defined herein.

The term “thioalkoxy” refers to an —SR′ group, wherein R′ is alkyl orcycloalkyl, as defined herein.

The term “thioaryloxy” refers to an —SR′ group, wherein R′ is aryl, asdefined herein.

The term “thioheteroaryloxy” refers to an —SR′ group, wherein R′ isheteroaryl, as defined herein.

The term “hydroxyalkyl,” as used herein, refers to an alkyl group, asdefined herein, substituted with one or more hydroxy group(s), e.g.,hydroxymethyl, 2-hydroxyethyl and 4-hydroxypentyl.

The term “aminoalkyl,” as used herein, refers to an alkyl group, asdefined herein, substituted with one or more amino group(s).

The term “alkoxyalkyl,” as used herein, refers to an alkyl groupsubstituted with one or more alkoxy group(s), e.g., methoxymethyl,2-methoxyethyl, 4-ethoxybutyl, n-propoxyethyl and t-butylethyl.

The term “trihaloalkyl” refers to —CX₃, wherein X is halo, as definedherein. An exemplary haloalkyl is CF₃.

A “guanidino” or “guanidine” or “guanidinyl” or “guanidyl” group refersto an —RaNC(═NRd)-NRbRc group, where each of Ra, Rb, Re and Rd can eachbe as defined herein for R′ and R″.

A “guanyl” or “guanine” group refers to an RaRbNC(═NRd)-group, where Ra,Rb and Rd are each as defined herein for R′ and R″.

In some of any of the embodiments described herein, the guanidine groupis —NH—C(═NH)—NH₂.

In some of any of the embodiments described herein, the guanyl group isH₂N—C(═NH)— group.

Any one of the amine (including modified amine), guanidine and guaninegroups described herein is presented as a free base form thereof, but ismeant to encompass an ionized form thereof at physiological pH, and/orwithin a salt thereof, e.g., a pharmaceutically acceptable salt thereof,as described herein.

Whenever an alkyl, cycloalkyl, aryl, alkaryl, heteroaryl,heteroalicyclic, acyl and any other moiety as described herein issubstituted, it includes one or more substituents, each canindependently be, but are not limited to, hydroxy, alkoxy, thiohydroxy,thioalkoxy, aryloxy, thioaryloxy, alkaryl, alkenyl, alkynyl, sulfonate,sulfoxide, thiosulfate, sulfate, sulfite, thiosulfite, phosphonate,cyano, nitro, azo, sulfonamide, carbonyl, thiocarbonyl, C-carboxylate,O-carboxylate, N-thiocarbamate, O-thiocarbamate, oxo, thiooxo, oxime,acyl, acyl halide, azo, azide, urea, thiourea, N-carbamate, O-carbamate,C-amide, N-amide, guanyl, guanidyl, hydrazine and hydrazide, as theseterms are defined herein.

The term “cyano” describes a —C≡N group.

The term “nitro” describes an —NO₂ group.

The term “sulfate” describes a —O—S(═O)₂—OR′ end group, as this term isdefined hereinabove, or an —O—S(═O)₂—O— linking group, as these phrasesare defined hereinabove, where R′ is as defined hereinabove.

The term “thiosulfate” describes a —O—S(═S)(═O)—OR′ end group or a—O—S(═S)(═O)—O— linking group, as these phrases are defined hereinabove,where R′ is as defined hereinabove.

The term “sulfite” describes an —O—S(═O)—O—R′ end group or a —O—S(═O)—O—group linking group, as these phrases are defined hereinabove, where R′is as defined hereinabove.

The term “thiosulfite” describes a —O—S(═S)—O—R′ end group or an—O—S(═S)—O— group linking group, as these phrases are definedhereinabove, where R′ is as defined hereinabove.

The term “sulfinate” describes a —S(═O)—OR′ end group or an —S(═O)—O—group linking group, as these phrases are defined hereinabove, where R′is as defined hereinabove.

The term “sulfoxide” or “sulfinyl” describes a —S(═O)R′ end group or an—S(═O)— linking group, as these phrases are defined hereinabove, whereR′ is as defined hereinabove.

The term “sulfonate” or “sulfonyl” describes a —S(═O)₂—R′ end group oran —S(═O)₂— linking group, as these phrases are defined hereinabove,where R′ is as defined herein.

The term “S-sulfonamide” describes a —S(═O)₂—NR′R″ end group or a—S(═O)₂—NR′— linking group, as these phrases are defined hereinabove,with R′ and R″ as defined herein.

The term “N-sulfonamide” describes an R'S(═O)₂—NR″— end group or a—S(═O)₂—NR′— linking group, as these phrases are defined hereinabove,where R′ and R″ are as defined herein.

The term “carbonyl” or “carbonate” as used herein, describes a —C(═O)—R′end group or a —C(═O)— linking group, as these phrases are definedhereinabove, with R′ as defined herein.

The term “thiocarbonyl” as used herein, describes a —C(═S)—R′ end groupor a —C(═S)— linking group, as these phrases are defined hereinabove,with R′ as defined herein.

The term “oxo” as used herein, describes a (═O) group, wherein an oxygenatom is linked by a double bond to the atom (e.g., carbon atom) at theindicated position.

The term “thiooxo” as used herein, describes a (═S) group, wherein asulfur atom is linked by a double bond to the atom (e.g., carbon atom)at the indicated position.

The term “oxime” describes a ═N—OH end group or a ═N—O— linking group,as these phrases are defined hereinabove.

The term “acyl halide” describes a —(C═O)R″″ group wherein R″ ″ ishalide, as defined hereinabove.

The term “azo” or “diazo” describes an —N═NR′ end group or an —N═N—linking group, as these phrases are defined hereinabove, with R′ asdefined hereinabove.

The term “azide” describes an —N₃ end group.

The term “carboxylate” as used herein encompasses C-carboxylate andO-carboxylate.

The term “C-carboxylate” describes a —C(═O)—OR′ end group or a —C(═O)—O—linking group, as these phrases are defined hereinabove, where R′ is asdefined herein.

The term “O-carboxylate” describes a —OC(═O)R′ end group or a—OC(═O)-linking group, as these phrases are defined hereinabove, whereR′ is as defined herein.

A carboxylate can be linear or cyclic. When cyclic, R′ and the carbonatom are linked together to form a ring, in C-carboxylate, and thisgroup is also referred to as lactone. Alternatively, R′ and O are linkedtogether to form a ring in O-carboxylate. Cyclic carboxylates canfunction as a linking group, for example, when an atom in the formedring is linked to another group.

The term “thiocarboxylate” as used herein encompasses C-thiocarboxylateand O-thiocarboxylate.

The term “C-thiocarboxylate” describes a —C(═S)—OR′ end group or a—C(═S)—O— linking group, as these phrases are defined hereinabove, whereR′ is as defined herein.

The term “O-thiocarboxylate” describes a —OC(═S)R′ end group or a—OC(═S)-linking group, as these phrases are defined hereinabove, whereR′ is as defined herein.

A thiocarboxylate can be linear or cyclic. When cyclic, R′ and thecarbon atom are linked together to form a ring, in C-thiocarboxylate,and this group is also referred to as thiolactone. Alternatively, R′ andO are linked together to form a ring in O-thiocarboxylate. Cyclicthiocarboxylates can function as a linking group, for example, when anatom in the formed ring is linked to another group.

The term “carbamate” as used herein encompasses N-carbamate andO-carbamate.

The term “N-carbamate” describes an R″OC(═O)—NR′— end group or a—OC(═O)—NR′— linking group, as these phrases are defined hereinabove,with R′ and R″ as defined herein.

The term “O-carbamate” describes an —OC(═O)—NR′R″ end group or an—OC(═O)—NR′— linking group, as these phrases are defined hereinabove,with R′ and R″ as defined herein.

A carbamate can be linear or cyclic. When cyclic, R′ and the carbon atomare linked together to form a ring, in O-carbamate. Alternatively, R′and O are linked together to form a ring in N-carbamate. Cycliccarbamates can function as a linking group, for example, when an atom inthe formed ring is linked to another group.

The term “carbamate” as used herein encompasses N-carbamate andO-carbamate.

The term “thiocarbamate” as used herein encompasses N-thiocarbamate andO-thiocarbamate.

The term “O-thiocarbamate” describes a —OC(═S)—NR′R″ end group or a—OC(═S)—NR′— linking group, as these phrases are defined hereinabove,with R′ and R″ as defined herein.

The term “N-thiocarbamate” describes an R″OC(═S)NR′— end group or a—OC(═S)NR′— linking group, as these phrases are defined hereinabove,with R′ and R″ as defined herein.

Thiocarbamates can be linear or cyclic, as described herein forcarbamates.

The term “dithiocarbamate” as used herein encompasses S-dithiocarbamateand N-dithiocarbamate.

The term “S-dithiocarbamate” describes a —SC(═S)—NR′R″ end group or a—SC(═S)NR′— linking group, as these phrases are defined hereinabove,with R′ and R″ as defined herein.

The term “N-dithiocarbamate” describes an R″SC(═S)NR′— end group or a—SC(═S)NR′— linking group, as these phrases are defined hereinabove,with R′ and R″ as defined herein.

The term “urea”, which is also referred to herein as “ureido”, describesa —NR′C(═O)—NR″R′″ end group or a —NR′C(═O)—NR″— linking group, as thesephrases are defined hereinabove, where R′ and R″ are as defined hereinand R′″ is as defined herein for R′ and R″.

The term “thiourea”, which is also referred to herein as “thioureido”,describes a —NR′—C(═S)—NR″R′″ end group or a —NR′—C(═S)—NR″— linkinggroup, with R′, R″ and R′″ as defined herein.

The term “amide” as used herein encompasses C-amide and N-amide.

The term “C-amide” describes a —C(═O)—NR′R″ end group or a—C(═O)—NR′-linking group, as these phrases are defined hereinabove,where R′ and R″ are as defined herein.

The term “N-amide” describes a R′C(═O)—NR″— end group or a R′C(═O)—N—linking group, as these phrases are defined hereinabove, where R′ and R″are as defined herein.

The term “hydrazine” describes a —NR′—NR″R′″ end group or a—NR′—NR″-linking group, as these phrases are defined hereinabove, withR′, R″, and R′″ as defined herein.

As used herein, the term “hydrazide” describes a —C(═O)—NR′—NR″R′″ endgroup or a —C(═O)—NR′—NR″— linking group, as these phrases are definedhereinabove, where R′, R″ and R′″ are as defined herein.

As used herein, the term “thiohydrazide” describes a —C(═S)—NR′—NR″R′″end group or a —C(═S)—NR′—NR″— linking group, as these phrases aredefined hereinabove, where R′, R″ and R′″ are as defined herein.

Processes:

Further according to embodiments of the present invention, there areprovided processes of preparing the compounds as described herein.

These processes are generally effected by providing a paromaminederivative and introducing thereto a desired modification to therebyobtain a pseudo-disaccharide compound as described herein.

Processes of preparing pseudo-trisaccharide compounds as describedherein are generally effected by devising appropriate acceptoraminoglycoside molecules and corresponding donor molecules, as is knownin the art of aminoglycosides.

Generally, the synthesis of pseudo-trisaccharide compounds according tosome embodiments of the present invention is accomplished using suitableacceptor and donor molecules and reaction conditions which allowreacting a protected derivative of the donor and of the acceptor andremoving the protecting group so as to obtain a desiredpseudo-trisaccharide of Formula Ia.

The term “acceptor” is used herein to describe the skeletal structurederived from paromamine which has an available (unprotected) hydroxylgroup at position C3′, C4′, C6 or C5, preferably C5, which is reactiveduring a glycosylation reaction, and can accept a glycosyl.

The term “donor” is used herein to describe the glycosyl that reactswith the acceptor to form the final pseudo-trisaccharide compound.

The term “glycosyl”, as used herein, refers to a chemical group which isobtained by removing the hydroxyl group from the hemiacetal function ofa monosaccharide.

The donors and acceptors are designed so as to form the desiredcompounds according to some embodiments of the present invention. Thefollowing describes some embodiments of this aspect of the presentinvention, presenting exemplary processes of preparing exemplary subsetsof the compounds described herein. More detailed processes of preparingexemplary compounds according to some embodiments of the presentinvention, are presented in the Examples section that follows below.

The syntheses of pseudo-trisaccharide compounds according to someembodiments of the present invention, generally include (i) preparing anacceptor compound by selective protection of one or more hydroxyls andamines at selected positions present on the paromamine scaffold, leavingthe selected position (e.g., C5) unprotected and therefore free toaccept a donor (glycosyl) compound as defined herein; (ii) preparing adonor compound by selective protection of one or more hydroxyls andamines at selected positions present on the glycosyl, leaving oneposition unprotected and therefore free to couple with an acceptorcompound as defined herein; (iii) subjecting the donor and the acceptorto a coupling reaction; and (iii) removing the protecting groups tothereby obtain the desired compound.

The phrase “protected group”, as used herein, refers to a group that issubstituted or modified so as to block its functionality and protect itfrom reacting with other groups under the reaction conditions (e.g., acoupling reaction as described herein). A protected group isre-generated by removal of the substituent or by being re-modified.

When an “amino-protected group” or “hydroxyl-protected group” are used,it is meant that a protecting group is attached or used to modify therespective group so as to generate the protected group.

The phrase “protecting group”, as used herein, refers to a substituentor a modification that is commonly employed to block or protect aparticular functionality while reacting other functional groups on thecompound. The protecting group is selected so as to release thesubstituent or to be re-modified, to thereby generate the desiredunprotected group.

For example, an “amino-protecting group” or “amine-protecting group” isa substituent attached to an amino group, or a modification of an aminogroup, that blocks or protects the amino functionality in the compound,and prevents it from participating in chemical reactions. Theamino-protecting group is removed by removal of the substituent or by amodification that re-generates an amine group.

Suitable amino-protected groups include azide (azido), N-phthalimido,N-acetyl, N-trifluoroacetyl, N-t-butoxycarbonyl (BOC),N-benzyloxycarbonyl (CBz) and N-9-fluorenylmethylenoxycarbonyl (Fmoc).

A “hydroxyl-protecting group” or “hydroxyl-protecting group” refers to asubstituent or a modification of a hydroxyl group that blocks orprotects the hydroxyl functionality, and prevents it from participatingin chemical reactions. The hydroxy-protecting group is removed byremoval of the substituent or by a modification that re-generates ahydroxy group.

Suitable hydroxy protected groups include isopropylidene ketal andcyclohexanone dimethyl ketal (forming a 1,3-dioxane with two adjacenthydroxyl groups), 4-methoxy-1-methylbenzene (forming a 1,3-dioxane withtwo adjacent hydroxyl groups), O-acetyl, O-chloroacetyl, O-benzoyl andO-silyl.

For a general description of protecting groups and their use, see T. W.Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, NewYork, 1991.

According to some embodiments, the amino-protected groups include anazido (N₃—) and/or an N-phthalimido group, and the hydroxyl-protectinggroups include O-acetyl (AcO—), O-benzoyl (BzO—) and/or O-chloroacetyl.

It is noted herein that when applicable, a “protected group” refers to amoiety in which one reactive function on a compound is protected or morethan one function are protected at the same time, such as in the case oftwo adjacent functionalities, e.g., two hydroxyl groups that can beprotected at once by a isopropylidene ketal.

In some embodiments, the donor compound is a protected monosaccharidewhich can be represented by the general Formula III:

In some embodiments, the donor compound is a protected monosaccharidewhich can be represented by the general Formula III, having a leavinggroup at position 1″ thereof, denoted L, and optionally a substituentR₁₂ at position 5″, as defined herein:

wherein:

L is a leaving group;

OT is a donor protected hydroxyl group;

R₁₂ is as defined herein for Formula Ib (the configuration at the 5″position as presented in Formula III being a non-limiting example); and

D is a protected or unprotected form of NR₁₄R₁₅ as defined for FormulaIb, wherein when R₁₄ and R₁₅ in Formula Ib are both hydrogen, D is adonor protected amine group.

As used herein, the phrase “leaving group” describes a labile atom,group or chemical moiety that readily undergoes detachment from anorganic molecule during a chemical reaction, while the detachment istypically facilitated by the relative stability of the leaving atom,group or moiety thereupon. Typically, any group that is the conjugatebase of a strong acid can act as a leaving group. Representativeexamples of suitable leaving groups according to some of the presentembodiments include, without limitation, trichloroacetimidate, acetate,tosylate, triflate, sulfonate, azide, halide, hydroxy, thiohydroxy,alkoxy, cyanate, thiocyanate, nitro and cyano.

According to some embodiments of the present invention, each of thedonor hydroxyl-protecting groups is O-benzoyl and the donoramino-protecting group in either R₁₅ or R₁₇ is azido, although otherprotecting groups are contemplated.

It is to be noted that when one of R₁₄ and R₁₅ is other than hydrogen,it can be protected or unprotected. Typically, when one of R₆ and R₇ isguanine or guanidine, a protecting group suitable for the reactionconditions (e.g., of a coupling reaction with an acceptor) can be used.Optionally, the guanine or guanidine are unprotected. When one of R₁₄and R₁₅ is an alkyl, aryl or cycloalkyl, typically D in Formula III isan unprotected form of NR₁₄R₁₅.

The structure of the donor compound sets the absolute structure of RingIII in the resulting compound according to some embodiments of thepresent invention, namely the stereo-configuration of the 5″ positionand the type of R₁₄, R₁₅ and R₁₂ in Formula Ib.

Exemplary acceptor molecules suitable for use in the preparation of thecompounds described herein, are represented by Formula IV:

wherein:

the dashed line represents an S-configuration or an R-configuration atposition 6′;

OP is an acceptor protected hydroxyl group;

AP is an acceptor protected amine group;

R₁ is as defined herein for Formula Ia or Ib;

A is an acceptor protected hydroxyl group (OP); or can otherwise be oneof the other groups defining OR₂, either protected or unprotected,according to the chemical nature of these groups and the reactionconditions; and

B is an acceptor protected amine group, in case R₇ is Formula Ia ishydrogen, or can otherwise be a protected or unprotected form of thegroups defining R₇.

According to some embodiments of the present invention, the acceptorhydroxyl-protected group is O-acetyl.

According to some embodiments of the present invention, the donoramino-protecting group is azido, although other protecting groups arecontemplated.

The acceptor hydroxyl-protected groups and the acceptor amino-protectedgroups at the various positions of the acceptors can be the same ordifferent each position.

In some embodiments, for example, in case R₇ is other than H, theacceptor is prepared by generating the moiety B, prior to reacting itwith the donor.

The structure of the acceptor compound sets the absolute structure ofRing I and Ring II in the resulting compound according to someembodiments of the present invention.

In some embodiments, the synthesis of pseudo-disaccharide compounds ofFormula Ia, according to some embodiments of the present invention, isaccomplished using an amino-protected compound of Formula V:

wherein:

the dashed line represents an S-configuration or an R-configuration atposition 6′;

AP is an acceptor protected amine group;

R₁ is as defined herein for Formula Ia;

A is an acceptor protected hydroxyl group (OP), as described herein; orcan otherwise be one of the other groups defining OR₂, either protectedor unprotected, according to the chemical nature of these groups and thereaction conditions.

Embodiments of the present invention further encompass any of theintermediate compounds described herein in the context of processes ofpreparing the compounds of the present embodiments.

Therapeutic Uses:

As known in the art, about a third of alleles causing genetic diseasescarry premature termination (stop) codons (PTCs), which lead to theproduction of truncated proteins. One possible therapeutic approachinvolves the induction and/or promotion of readthrough of such PTCs toenable synthesis of full-length proteins. PTCs originate from eithermutations, such as nonsense mutations, frame-shift deletions andinsertions, or from aberrant splicing that generates mRNA isoforms withtruncated reading frames. These mutations can lead to the production oftruncated, nonfunctional or deleterious proteins, owing to dominantnegative or gain-of-function effects.

In general, readthrough of PTCs can be achieved by suppressor transferRNAs (tRNAs), factors that decrease translation-termination efficiency,such as small-interfering RNAs (siRNAs) directed against thetranslation-termination factors, and RNA antisense that targets thenonsense mutation region. One of the objectives of the present inventionis to provide a pharmacological therapeutic approach aimed at achievingsufficient levels of functional proteins in a subject suffering from atleast one genetic disorder associated with at least one prematurestop-codon mutation. According to embodiments of the present invention,the provided therapeutic approach is aimed at inducing and/or promotingtranslational readthrough of the disease causing PTCs, to enable thesynthesis and expression of full-length functional proteins.

As presented hereinabove, one extensively studied approach that hasreached clinical trials, is based on readthrough by drugs affecting theribosome decoding site, such as aminoglycoside antibiotics; however,aminoglycosides have severe adverse side effects when used at highconcentrations and/or used long-term. The compounds presented hereinwere designed to exhibit an ability to induce and/or promote readthroughof a premature stop-codon mutation in an organism having such amutation, while exhibiting minimal adverse effects. Such an activityrenders these compounds suitable for use as therapeutically activeagents for the treatment of genetic disorders associated with apremature stop-codon mutation.

As demonstrated in the Examples section that follows, exemplarycompounds encompassed by the present embodiments were indeed shown toexhibit a premature stop-codon mutation suppression activity, and henceas useful in inducing readthrough of genes characterized by adisease-causing premature stop-codon mutation, and thus exhibitusefulness in treating respective genetic diseases or disordersassociated with a premature stop-codon mutation.

According to an aspect of some embodiments of the present invention, anyof the compounds presented herein having Formula Ia or Ib, including anyof the respective embodiments of the compounds and any combinationsthereof (and including compounds represented by Formula IC, Id and Ie),are for use in inducing and/or promoting readthrough of a premature stopcodon mutation and/or for increasing an expression of a gene having apremature stop codon mutation, and/or are for use in the manufacture ofa medicament for inducing and/or promoting readthrough of a prematurestop codon mutation and/or for increasing an expression of a gene havinga premature stop codon mutation.

According to an aspect of some embodiments of the present invention, anyof the compounds presented herein having Formula Ia or Ib, including anyof the respective embodiments of the compounds and any combinationsthereof (and including compounds represented by Formula IC, Id and Ie),are for use in the treatment of a genetic disorder associated with apremature stop-codon mutation, or for use in the manufacture of amedicament for the treatment of a genetic disorder associated with apremature stop-codon mutation.

Any of the premature stop-codon mutations are contemplated. In someembodiments, the mutations are those having an RNA code of UGA, UAG orUAA.

According to some of any of the embodiments described herein, theprotein is translated in a cytoplasmic translation system.

According to some of any of the embodiments described herein, thecompound is used in a mutation suppression amount.

According to some of any of the embodiments described herein, aninhibition of translation IC₅₀ of the compound in a eukaryoticcytoplasmic translation system is greater that an inhibition oftranslation IC₅₀ of the compound in a ribosomal translation system.

According to some of any of the embodiments described herein, aninhibition of translation IC₅₀ of the compound in a eukaryoticcytoplasmic translation system is greater that an inhibition oftranslation IC₅₀ of the compound in a prokaryotic translation system.

According to an aspect of some embodiments of the present invention, anyof the compounds presented herein having Formula Ia or Ib, including anyof the respective embodiments of the compounds and any combinationsthereof (and including compounds represented by Formula IC, Id and Ie),are for use in the treatment of a genetic disorder associated with apremature stop-codon mutation, or for use in the manufacture of amedicament for the treatment of a genetic disorder associated with apremature stop-codon mutation.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating a genetic disorder associatedwith a premature stop-codon mutation. The method, according to thisaspect of the present invention, is effected by administering to asubject in need thereof a therapeutically effective amount of one ormore of the compounds presented herein having Formula Ia or Ib,including any of the respective embodiments of the compounds and anycombinations thereof (and including compounds represented by Formula IC,Id and Ie).

As used herein, the term “treating” includes abrogating, substantiallyinhibiting, slowing or reversing the progression of a condition,substantially ameliorating clinical or aesthetical symptoms of acondition or substantially preventing the appearance of clinical oraesthetical symptoms of a condition.

As used herein, the phrase “therapeutically effective amount” describesan amount of the polymer being administered which will relieve to someextent one or more of the symptoms of the condition being treated.

The phrase “genetic disorder”, as used herein, refers to a chronicdisorder which is caused by one or more defective genes that are ofteninherited from the parents, and which can occur unexpectedly when twohealthy carriers of a defective recessive gene reproduce, or when thedefective gene is dominant. Genetic disorders can occur in differentinheritance patterns which include the autosomal dominant patternwherein only one mutated copy of the gene is needed for an offspring tobe affected, and the autosomal recessive pattern wherein two copies ofthe gene must be mutated for an offspring to be affected.

The phrase “genetic disorder”, as used herein, encompasses a geneticdisorder, genetic disease, genetic condition or genetic syndrome.

According to some of any of the embodiments of the present invention,the genetic disorder, genetic disease, genetic condition or geneticsyndrome, involves a gene having a premature stop-codon mutation, alsoreferred to herein as a truncation mutation and/or a nonsense mutation,which leads to improper translation thereof. The improper translationproduces a dysfunctional essential protein or causes a reduction orabolishment of synthesis of an essential protein. In the context of thesome embodiments of the present invention, the genetic disorders whichare contemplated within the scope of the present embodiments arereferred to as genetic disorders associated with a premature stop-codonmutation and/or a protein truncation phenotype.

According to some of any of the embodiments of the present invention, agenetic disorder associated with a premature stop-codon mutation and/ora protein truncation phenotype is treatable by inducing and/or promotingreadthrough of the mutation in the complete but otherwise defectivetranscript (mRNA), or in other words, by inducing and/or promotingsuppression of the nonsense mutation (the premature stop-codon mutationand/or the truncation mutation). In the context of embodiments of thepresent invention, a genetic disorder is one that is treatable byreadthrough-inducing and/or promoting compounds.

Methods for identification of a genetic disorder associated with apremature stop-codon mutation and/or a protein truncation phenotype arewell known in the art, and include full or partial genome elucidation,genetic biomarker detection, phenotype classification and hereditaryinformation analysis.

Such methods often result in pairs of mutant/wild type (WT) sequences,and these pairs can be used in known methodologies for identifying ifthe genetic disorder is associated with a premature stop-codon mutationand/or a protein truncation phenotype.

A readthrough-inducing/promoting activity of compounds for treating suchgenetic disorders can be established by methods well known in the art.

For example, a plasmid comprising two reporter genes interrupted by asequence of the mutated gene (the genetic disorder-causing gene) istransected into a protein expression platform, either in full cells orin a cell-free systems, and the ratio between the expression level ofthe two genes in the presence of a tested compound is measured,typically in series of concentrations and duplications, and compared tothe gene expression level ratio of the wild-type and/or to theexpression level ratio measured in a control sample not containing thetested compound.

It is noted that the experimental model for readthrough activity, namelythe nucleotide sequence of gene containing the premature stop-codonmutation, is a byproduct of the process of identifying a geneticdisorder as associated with a premature stop-codon mutation and/or aprotein truncation phenotype, and further noted that with the greatadvances in genomic data acquisition, this process is now well withinthe skills of the artisans of the art, and that once the mechanism ofaction of a drug candidate is established, as in the case of geneticdisorders which have been shown to be associated with a prematurestop-codon mutation and/or a protein truncation phenotype, it is wellwithin the skills of the artisans of the art to identify, characterizeand assess the efficacy, selectivity and safety of any one of thereadthrough-inducing compounds presented herein. It is further wellwithin the skills of the artisans of the art to take thereadthrough-inducing compounds presented herein further though theroutine processes of drug development.

Methodologies for testing readthrough of a premature stop-codon mutationand/or a truncation mutation, referred to herein as readthroughactivity, are known in the art, and several exemplary experimentalmethods are provided in the Examples section that follows, by which thereadthrough-inducing compounds, according to some embodiments of thepresent invention, can be characterized. It is to be understood thatother methods can be used to characterized readthrough-inducingcompounds, and such methods are also contemplated within the scope ofthe present invention. Methods such as provided herein can also beadapted for high throughput screening technology that can assaythousands of compounds in a relatively short period of time.

The skilled artisan would appreciate that many in vitro methodologiescan be used to characterize readthrough-inducing compounds providedherein in terms of safety of use as drugs, and assess the drugcandidates in terms of their cytotoxicity versus their efficacy. Theskilled artisan would also appreciate that many in vitro methodologiescan be used to characterize the readthrough-inducing compounds providedherein for eukaryotic versus prokaryotic selectivity, and suchmethodologies may also be adapted for high throughput screeningtechnology that can assay thousands of compounds in a relatively shortperiod of time.

Non-limiting examples of genetic disorders, diseases, conditions andsyndromes, which are associated with the presence of at least onepremature stop-codon or other nonsense mutations include cancer, Rettsyndrome, cystic fibrosis (CF), Becker's muscular dystrophy (BMD),Congenital muscular dystrophy (CMD), Duchenne muscular dystrophy (DMD),Factor VII deficiency, Familial atrial fibrillation, Hailey-Haileydisease, hemophilia A, hemophilia B, Hurler syndrome, Louis-Bar syndrome(ataxia-telangiectasia, AT), McArdle disease, Mucopolysaccharidosis,Nephropathic cystinosis, Polycystic kidney disease, type I, II and IIISpinal muscular atrophy (SMA), Tay-Sachs, Usher syndrome, cystinosis,Severe epidermolysis bullosa, Dravet syndrome, X-linked nephrogenicdiabetes insipidus (XNDI) and X-linked retinitis pigmentosa.

Additional genetic disorders, diseases, conditions and syndromes, whichare associated with the presence of at least one premature stop-codon orother nonsense mutations, are listed in “Suppression of nonsensemutations as a therapeutic approach to treat genetic diseases” by Kim M.Keeling, K. M Bedwell, D. M., Wiley Interdisciplinary Reviews: RNA,2011, 2(6), p. 837-852; “Cancer syndromes and therapy by stop-codonreadthrough”, by Bordeira-Carrico, R. et al., Trends in MolecularMedicine, 2012, 18(11), p. 667-678, and any references cited therein,all of which are incorporated herewith by reference in their entirety.

In any of the methods and uses described herein, the compounds describedherein can be utilized either per se or form a part of a pharmaceuticalcomposition, which further comprises a pharmaceutically acceptablecarrier, as defined herein.

According to an aspect of some embodiments of the present invention,there is provided a pharmaceutical composition which comprises, as anactive ingredient, any of the novel compounds described herein and apharmaceutically acceptable carrier.

As used herein a “pharmaceutical composition” refers to a preparation ofthe compounds presented herein, with other chemical components such aspharmaceutically acceptable and suitable carriers and excipients. Thepurpose of a pharmaceutical composition is to facilitate administrationof a compound to an organism.

Hereinafter, the term “pharmaceutically acceptable carrier” refers to acarrier or a diluent that does not cause significant irritation to anorganism and does not abrogate the biological activity and properties ofthe administered compound. Examples, without limitations, of carriersare: propylene glycol, saline, emulsions and mixtures of organicsolvents with water, as well as solid (e.g., powdered) and gaseouscarriers.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of acompound. Examples, without limitation, of excipients include calciumcarbonate, calcium phosphate, various sugars and types of starch,cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore pharmaceutically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the compounds presentedherein into preparations which, can be used pharmaceutically. Properformulation is dependent upon the route of administration chosen.

According to some embodiments, the administration is effected orally.For oral administration, the compounds presented herein can beformulated readily by combining the compounds with pharmaceuticallyacceptable carriers well known in the art. Such carriers enable thecompounds presented herein to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for oral ingestion by a patient. Pharmacological preparations for oraluse can be made using a solid excipient, optionally grinding theresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/orphysiologically acceptable polymers such as polyvinylpyrrolidone (PVP).If desired, disintegrating agents may be added, such as cross-linkedpolyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such assodium alginate.

Pharmaceutical compositions, which can be used orally, include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, thecompounds presented herein may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for the chosen routeof administration.

For injection, the compounds presented herein may be formulated inaqueous solutions, preferably in physiologically compatible buffers suchas Hank's solution, Ringer's solution, or physiological saline bufferwith or without organic solvents such as propylene glycol, polyethyleneglycol.

For transmucosal administration, penetrants are used in the formulation.Such penetrants are generally known in the art.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active aminoglycoside compounds doses.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds presented herein areconveniently delivered in the form of an aerosol spray presentation(which typically includes powdered, liquefied and/or gaseous carriers)from a pressurized pack or a nebulizer, with the use of a suitablepropellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,dichloro-tetrafluoroethane or carbon dioxide. In the case of apressurized aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compounds presented herein and a suitablepowder base such as, but not limited to, lactose or starch.

The compounds presented herein may be formulated for parenteraladministration, e.g., by bolus injection or continuous infusion.Formulations for injection may be presented in unit dosage form, e.g.,in ampoules or in multidose containers with optionally, an addedpreservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the compounds preparation in water-soluble form.Additionally, suspensions of the compounds presented herein may beprepared as appropriate oily injection suspensions and emulsions (e.g.,water-in-oil, oil-in-water or water-in-oil in oil emulsions). Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acids esters such as ethyl oleate, triglycerides orliposomes. Aqueous injection suspensions may contain substances, whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents, which increase the solubility ofthe compounds presented herein to allow for the preparation of highlyconcentrated solutions.

Alternatively, the compounds presented herein may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free water,before use.

The compounds presented herein may also be formulated in rectalcompositions such as suppositories or retention enemas, using, e.g.,conventional suppository bases such as cocoa butter or other glycerides.

The pharmaceutical compositions herein described may also comprisesuitable solid of gel phase carriers or excipients. Examples of suchcarriers or excipients include, but are not limited to, calciumcarbonate, calcium phosphate, various sugars, starches, cellulosederivatives, gelatin and polymers such as polyethylene glycols.

Pharmaceutical compositions suitable for use in context of the presentinvention include compositions wherein the active ingredients arecontained in an amount effective to achieve the intended purpose. Morespecifically, a therapeutically effective amount means an amount ofcompounds presented herein effective to prevent, alleviate or amelioratesymptoms of the disorder, or prolong the survival of the subject beingtreated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any compounds presented herein used in the methods of the presentembodiments, the therapeutically effective amount or dose can beestimated initially from activity assays in animals. For example, a dosecan be formulated in animal models to achieve a circulatingconcentration range that includes the mutation suppression levels asdetermined by activity assays (e.g., the concentration of the testcompounds which achieves a substantial read-through of the truncationmutation). Such information can be used to more accurately determineuseful doses in humans.

Toxicity and therapeutic efficacy of the compounds presented herein canbe determined by standard pharmaceutical procedures in experimentalanimals, e.g., by determining the EC₅₀ (the concentration of a compoundwhere 50% of its maximal effect is observed) and the LD₅₀ (lethal dosecausing death in 50% of the tested animals) for a subject compound. Thedata obtained from these activity assays and animal studies can be usedin formulating a range of dosage for use in human.

The dosage may vary depending upon the dosage form employed and theroute of administration utilized. The exact formulation, route ofadministration and dosage can be chosen by the individual physician inview of the patient's condition. (See e.g., Fingl et al., 1975, in “ThePharmacological Basis of Therapeutics”, Ch. 1 p. 1).

Dosage amount and interval may be adjusted individually to provideplasma levels of the compounds presented herein which are sufficient tomaintain the desired effects, termed the minimal effective concentration(MEC). The MEC will vary for each preparation, but can be estimated fromin vitro data; e.g., the concentration of the compounds necessary toachieve 50-90% expression of the whole gene having a truncationmutation, i.e. read-through of the mutation codon. Dosages necessary toachieve the MEC will depend on individual characteristics and route ofadministration. HPLC assays or bioassays can be used to determine plasmaconcentrations.

Dosage intervals can also be determined using the MEC value.Preparations should be administered using a regimen, which maintainsplasma levels above the MEC for 10-90% of the time, preferable between30-90% and most preferably 50-90%.

Depending on the severity and responsiveness of the chronic condition tobe treated, dosing can also be a single periodic administration of aslow release composition described hereinabove, with course of periodictreatment lasting from several days to several weeks or until sufficientamelioration is effected during the periodic treatment or substantialdiminution of the disorder state is achieved for the periodic treatment.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc. Compositions of the present invention may, if desired, be presentedin a pack or dispenser device, such as an FDA (the U.S. Food and DrugAdministration) approved kit, which may contain one or more unit dosageforms containing the active ingredient. The pack may, for example,comprise metal or plastic foil, such as, but not limited to a blisterpack or a pressurized container (for inhalation). The pack or dispenserdevice may be accompanied by instructions for administration. The packor dispenser may also be accompanied by a notice associated with thecontainer in a form prescribed by a governmental agency regulating themanufacture, use or sale of pharmaceuticals, which notice is reflectiveof approval by the agency of the form of the compositions for human orveterinary administration. Such notice, for example, may be of labelingapproved by the U.S. Food and Drug Administration for prescription drugsor of an approved product insert. Compositions comprising a compoundaccording to the present embodiments, formulated in a compatiblepharmaceutical carrier may also be prepared, placed in an appropriatecontainer, and labeled for treatment of an indicated condition ordiagnosis, as is detailed hereinabove.

Thus, in some embodiments, the pharmaceutical composition is packaged ina packaging material and identified in print, in or on the packagingmaterial, for use in the treatment of a genetic disorder, as definedherein, and/or in any of the uses described herein.

In some embodiments, the pharmaceutical composition is for use in thetreatment of a genetic disorder, as defined herein, and/or in any of theuses described herein.

In any of the composition, methods and uses described herein, thecompounds can be utilized in combination with other agents useful in thetreatment of the genetic disorder and/or in inducing or promotingreadthrough activity of a premature stop codon mutation and/or inincreasing expression of a gene having a premature stop codon mutationas described herein.

Being primarily directed at treating genetic disorders, which arechronic by definition, the compounds presented herein or pharmaceuticalcompositions containing the same are expected to be administeredthroughout the lifetime of the subject being treated. Therefore, themode of administration of pharmaceutical compositions containing thecompounds should be such that will be easy and comfortable foradministration, preferably by self-administration, and such that willtake the smallest toll on the patient's wellbeing and course of life.

The repetitive and periodic administration of the compounds presentedherein or the pharmaceutical compositions containing the same can beeffected, for example, on a daily basis, i.e. once a day, morepreferably the administration is effected on a weekly basis, i.e. once aweek, more preferably the administration is effected on a monthly basis,i.e. once a month, and most preferably the administration is effectedonce every several months (e.g., every 1.5 months, 2 months, 3 months, 4months, 5 months, or even 6 months).

As discussed hereinabove, some of the limitations for using presentlyknown aminoglycosides as truncation mutation readthrough drugs areassociated with the fact that they are primarily antibacterial (used asantibiotic agents). Chronic use of any antibacterial agents is highlyunwarranted and even life threatening as it alters intestinal microbialflora which may cause or worsen other medical conditions such as flaringof inflammatory bowel disease, and may cause the emergence of resistancein some pathological strains of microorganisms.

In some embodiments, the compounds presented herein have substantiallyno antibacterial activity. By “no antibacterial activity” it is meantthat the minimal inhibition concentration (MIC) thereof for a particularstrain is much higher than the concentration of a compound that isconsidered an antibiotic with respect to this strain. Further, the MICof these compounds is notably higher than the concentration required forexerting truncation mutation suppression activity.

Being substantially non-bactericidal, the compounds presented herein donot exert the aforementioned adverse effects and hence can beadministered via absorption paths that may contain benign and/orbeneficial microorganisms that are not targeted and thus theirpreservation may even be required. This important characteristic of thecompounds presented herein renders these compounds particularlyeffective drugs against chronic conditions since they can beadministered repetitively and during life time, without causing anyantibacterial-related adverse, accumulating effects, and can further beadministered orally or rectally, i.e. via the GI tract, which is a veryhelpful and important characteristic for a drug directed at treatingchronic disorders.

According to some embodiments, the compounds presented herein areselected and/or designed to be selective towards the eukaryotic cellulartranslation system versus that of prokaryotic cells, namely thecompounds exhibit higher activity in eukaryotic cells, such as those ofmammalian (humans) as compared to their activity in prokaryotic cells,such as those of bacteria. Without being bound by any particular theory,it is assumed that the compounds presented herein, which are known toact by binding to the A-site of the 16S ribosomal RNA while the ribosomeis involved in translating a gene, have a higher affinity to theeukaryotic ribosomal A-site, or otherwise are selective towards theeukaryotic A-site, versus the prokaryotic ribosomal A-site, as well asthe mitochondrial ribosomal A-site which resembles its prokaryoticcounterpart.

As used herein the term “about” refers to ±10%.

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”.

The term “consisting of” means “including and limited to”.

The term “consisting essentially of” means that the composition, methodor structure may include additional ingredients, steps and/or parts, butonly if the additional ingredients, steps and/or parts do not materiallyalter the basic and novel characteristics of the claimed composition,method or structure.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof. Throughout this application,various embodiments of this invention may be presented in a rangeformat. It should be understood that the description in range format ismerely for convenience and brevity and should not be construed as aninflexible limitation on the scope of the invention. Accordingly, thedescription of a range should be considered to have specificallydisclosed all the possible subranges as well as individual numericalvalues within that range. For example, description of a range such asfrom 1 to 6 should be considered to have specifically disclosedsubranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4,from 2 to 6, from 3 to 6 etc., as well as individual numbers within thatrange, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of thebreadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

It is expected that during the life of a patent maturing from thisapplication many relevant genetic diseases and disorders as definedherein will be uncovered and the scope of this term is intended toinclude all such new disorders and diseases a priori.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions illustrate some embodiments of the invention in a nonlimiting fashion.

Example 1 Chemical Syntheses of Cell-Permealizing Group-ContainingExemplary Compounds According to Some of the Present Embodiments

In general, aminoglycosides (AGs) antibiotic are charged atphysiological pH, thus they may be limited in their absorption throughthe GI tract and are therefore typically administered by injection. Inaddition, AGs exhibit limited permeability into eukaryotic cells, whichrequires their administration in higher dosages in order to overcome thecellular uptake limitation, which in turn causes adverse effects andlimits their use in translational therapy. The compounds described inthis example were designed in order to solve these problems.

To mitigate the GI tract absorption problem, alkyl/aryl groups have beenattached on the pseudo-disaccharide scaffold at the N1 position of aparomamine-derived aminoglycoside. Exemplary compounds NB144, NB145,NB146 and NB147 (see, Table 1 herein), were prepared so as to exhibitrespectively an isopropyl, a benzyl, a propyl and a propyl substitutionat the N-1 position.

To mitigate the cellular uptake limitation, a series of compounds wasprepared with cell-permealizable groups so as to increase their cellularuptake. These compounds were prepared by introducing acell-permealizable group, such as a guanidine group, at variouspositions on the scaffold.

The following are processes for preparing exemplary compounds accordingto some embodiments of the present invention, which are presented inTable 1 hereinabove.

The synthesis of compounds NB144, NB145 and NB146 was accomplished intwo steps starting with Compound 1, (prepared as previously reported inBaasov et al., Bioorg. Med. Chem., 2010, 18, pp. 3735-3746), asillustrated in a general Scheme 2 below (reagents and conditions: (i)RCHO, H₂O, 1M HCl, NaBCNH₃ or RCHO, MeOH, NaBH₄ 0° C.; (ii) PMe3, NaOH,THF, room temperature).

Monoalkylation of primary amine with aliphatic aldehydes was performedin water with sodium borocyanohydride, while methanol/NaBH₄ was used inthe case of benzaldehyde. The total yield of this step was 35-58% ofmonoalkylated/benzylated products 2a-c (Scheme 2). The Staudingerreaction was then performed to obtain the final compounds NB144, NB145and NB146 in good yields of 68-85%.

Synthesis of NB144

NB144 was prepared according to Scheme 2 hereinabove, starting with theprecursor Compound 1. Compound 1 (0.5 grams, 1.2 mmol) was dissolved andstirred in water (15 mL) at 0° C. for 15 minutes, and 1 M solution ofhydrochloric acid was added dropwise to adjust the pH of the reactionmixture to about 2-3. About 2 equivalents of isobutyraldehyde (0.2 mL)were added to the reaction mixture and stirred for 15 minutes at roomtemperature. The resulted solution was cooled to 0° C. and NaBCNH₃ (30mg, 1.5 equivalents) was added and progress was monitored by TLC. After1 hour of reaction, the similar process was repeated until startingmaterial was consumed to desired product. After completion, the reactionmixture was evaporated and subjected to column chromatography to obtainthe mono alkylated product, Compound 2a (0.2 grams, 35%). Compound 2awas dissolved in a mixture of THF (5 mL) and aqueous NaOH (1 mM, 5.0mL). The mixture was stirred at room temperature for 10 minutes, afterwhich PMe₃ (1 M solution in THF, 2.0 mL, 2.0 mmol) was added. Thereaction progress was monitored by TLC [CH₂Cl₂/MeOH/H₂O/MeNH₂ (33%solution in EtOH) 10:15:6:15], which indicated completion after 1 hour.The product was purified by column chromatography on a short column ofsilica gel. The column was washed with the following solvents: THF (200mL), CH₂Cl₂ (200 mL), EtOAc (100 mL), and MeOH (200 mL). The product wasthen eluted with a mixture of MeNH₂ (33% solution in EtOH) and MeOH(8:2). Fractions containing the product were combined and evaporated todryness. The residue was re-dissolved in a small volume of water andevaporated again (2-3 repeats) to afford the free amine form of NB144.The analytically pure product was obtained by passing the above productthrough a short column of Amberlite CG50 (NH₄ ⁺ form). The column wasfirst washed with a mixture of MeOH/H₂O (3:2), then the product waseluted with a mixture of MeOH/H₂O/NH₄OH (8:1:1) to afford title compoundNB144 (0.150 grams, 85%). For the storage and biological tests, NB144was converted to its sulfate salt form: the free base was dissolved inwater, the pH was adjusted around 7.0 with H₂SO₄ (0.1 N) andlyophilized.

¹HNMR (500 MHz, CD₃OD): “Ring I”: δ_(H)=1.21 (d, 3H, J=6.0 Hz, CH₃),2.70 (dd, 1H, J₁=3.4, J₂=10.0 Hz, H-2′), 3.21 (t, 1H, J=10.0 Hz, H-4′),3.48 (t, 1H, J=9.0 Hz, H-3′), 3.81 (dd, 1H, J₁=3.4, J₂=10.0 Hz, H-5′),4.09 (m, 1H, H-6′), 5.16 (d, 1H, J=2.5 Hz, H-1′); “Ring II”: δ_(H)=1.11(m, 1H, H-2_(ax)), 2.14 (td, 1H, J₁=4.5, J₂=12.5 Hz, H-2_(eq)), 2.46 (m,1H, H-1), 2.71 (m, 1H, H-3), 3.19 (m, 2H, H-4 and H-6), 3.44 (t, 1H,J=9.1 Hz, H-5). The additional peaks in the spectrum were identified asfollows: δ_(H)=0.96 (t, 3H, J=3.1 Hz), 0.97 (t, 3H, J=3.2 Hz), 1.79 (m,1H), 2.32 (m, 1H), 2.56 (m, 1H).

¹³CNMR (125 MHz, CD₃OD): δ₆=16.6, 20.8, 20.9, 29.0, 34.6, 51.5, 55.8,57.4, 58.9, 67.8, 73.6, 75.8, 76.5 (2C), 77.8, 90.9, 103.2 (C-1′).

MALDI TOFMS: calculated for C₁₇H₃₆N₃O₇ ([M+H]⁺) m/e: 394.2; measuredm/e: 394.1.

Synthesis of NB145

NB145 was prepared according to Scheme 2 presented hereinabove, startingwith the precursor Compound 1. Compound 1 (0.5 grams, 1.2 mmol) andbenzaldehyde (0.3 grams 4 mmol) were dissolved and stirred in methanol(15 mL) at room temperature for 15 minutes. The resulted solution wascooled to 0° C. and NaBH₄ (100 mg) was added and progress was monitoredby TLC. After completion, the reaction mixture evaporated and subjectedto column chromatography to obtain the mono benzylated Compound 2b in0.3 grams, 50% yield. Compound 2b was dissolved in a mixture of THF (5mL) and aqueous NaOH (1 mM, 5.0 mL). The mixture was stirred at roomtemperature for 10 minutes, after which PMe₃ (1 M solution in THF, 2.0mL, 2.0 mmol) was added. The reaction progress was monitored by TLC[CH₂Cl₂/MeOH/H₂O/MeNH₂ (33% solution in EtOH) 10:15:6:15], whichindicated completion after 1 hour. The product was purified by columnchromatography on a short column of silica gel. The column was washedwith the following solvents: THF (200 mL), CH₂Cl₂ (200 mL), EtOAc (100mL), and MeOH (200 mL). The product was then eluted with a mixture ofMeNH₂ (33% solution in EtOH) and MeOH (8:2). Fractions containing theproduct were combined and evaporated to dryness. The residue wasre-dissolved in a small volume of water and evaporated again (2-3repeats) to afford the free amine form of NB145. The analytically pureproduct was obtained by passing the above product through a short columnof Amberlite CG50 (NH₄ ⁺ form). The column was first washed with amixture of MeOH/H₂O (3:2), then the product was eluted with a mixture ofMeOH/H₂O/NH₄OH (8:1:1) to afford NB145 (0.200 grams, 75% yield). For thestorage and biological tests, NB145 was converted to its sulfate saltform: the free base was dissolved in water, the pH was adjusted around7.0 with H₂SO₄ (0.1 N) and lyophilized.

¹HNMR (500 MHz, CD₃OD): “Ring I”: δ_(H)=1.21 (d, 3H, J=6.0 Hz, CH₃),2.73 (dd, 1H, J₁=4.6, J₂=10.3 Hz, H-2′), 3.23 (t, 1H, J=10.0 Hz, H-4′),3.49 (t, 1H, J=9.0 Hz, H-3′), 3.82 (dd, 1H, J₁=3.4, J₂=10.0 Hz, H-5′),4.12 (m, 1H, H-6′), 5.18 (d, 1H, J=2.5 Hz, H-1′); “Ring II”: δ_(H)=1.15(m, 1H, H-2_(ax)), 2.23 (td, 1H, J₁=4.5, J₂=12.5 Hz, H-2_(eq)), 2.56 (m,1H, H-1), 2.70 (m, 1H, H-3), 3.22 (t, 1H, J=9.2 Hz, H-6), 3.28 (t, 1H,J=9.0 Hz, H-4), 3.43 (t, 1H, J=9.1 Hz, H-5). The additional peaks in thespectrum were identified as follows: δ_(H)=3.65 (d, 1H, J=12.5 Hz), 3.92(d, 1H, J=12.5 Hz), 7.28-7.37 (m, 5H, Ar).

¹³CNMR (125 MHz, CD₃OD): δ_(C)=16.4, 34.2, 51.1, 51.6, 57.2, 57.8, 67.6,73.2, 75.7, 76.3, 76.4, 77.7, 90.2, 102.9 (C-1′), 128.3 (Ar), 129.4(Ar), 129.6 (Ar), 140.3 (Ar).

MALDI TOFMS calculated for C₂₀H₃₄N₃O₇ ([M+H]⁺) m/e: 428.2; measured m/e:428.1.

Synthesis of NB146

NB146 was prepared according to Scheme 2 presented hereinabove, startingwith Compound 1. Compound 1 (0.5 grams, 1.2 mmol) was dissolved andstirred in water (15 mL) at 0° C. for 15 minutes and a 1 M solution ofhydrochloric acid was added dropwise to adjust the pH of the reactionmixture to about 2-3. About 2 equivalents of propyl aldehyde (0.2 mL)were added to the reaction mixture and stirred for 15 minutes at roomtemperature. The resulted solution was cooled to 0° C. and NaBCNH₃ (30mg, 1.5 equivalents) was added and progress was monitored by TLC. After1 hour of reaction, the similar process was repeated until startingmaterial was consumed to desired product. After completion, the reactionmixture was evaporated and subjected to column chromatography to obtainCompound 2c in 0.325 g (58%).

¹HNMR (500 MHz, CD₃OD): “Ring I”: δ_(H)=1.27 (d, 3H, J=6.0 Hz, CH₃),3.09 (dd, 1H, J₁=4.2, J₂=10.5 Hz, H-2′), 3.39 (dd, 1H, J₁=8.7, J₂=10.0Hz, H-4′), 3.94 (m, 2H, H-3′ and H-5′), 4.04 (m, 1H, H-6′), 5.73 (d, 1H,J=3.5 Hz, H-1′); “Ring II”: δ_(H)=1.26 (m, 1H, H-2_(ax)), 2.31 (td, 1H,J₁=4.5, J₂=12.5 Hz, H-2_(eq)), 2.54 (m, 1H, H-1), 3.15 (m, 1H, H-3),3.46-3.54 (m, 3H, H-4, H-5 and H-6). The additional peaks in thespectrum were identified as follows: δ_(H)=0.98 (t, 3H, J=7.2 Hz), 1.56(m, 2H), 2.53 (m, 1H), 2.72 (m, 1H).

¹³CNMR (125 MHz, CD₃OD): δ_(C)=11.9, 18.1, 23.6, 32.6 (C-2), 49.7, 57.9,61.7, 64.7, 69.4, 72.3, 74.3, 75.2, 76.7, 78.6, 80.7, 98.6 (C-1′).

MALDI TOFMS calculated for C₁₆H₃₀N₇O₇([M+H]⁺) m/e: 432.2; measured m/e:432.2.

Compound 2c (0.325 grams, 0.75 mmol) was dissolved in a mixture of THF(5 mL) and aqueous NaOH (1 mM, 5.0 mL). The mixture was stirred at roomtemperature for 10 minutes, after which PMe₃ (1 M solution in THF, 2.0mL, 2.0 mmol) was added. The reaction progress was monitored by TLC[CH₂Cl₂/MeOH/H₂O/MeNH₂ (33% solution in EtOH) 10:15:6:15], whichindicated completion after 1 hour. The product was purified by columnchromatography on a short column of silica gel. The column was washedwith the following solvents: THF (200 mL), CH₂Cl₂ (200 mL), EtOAc (100mL), and MeOH (200 mL). The product was then eluted with a mixture of20% MeNH₂ (33% solution in EtOH) in 80% MeOH. Fractions containing theproduct were combined and evaporated to dryness. The residue wasre-dissolved in a small volume of water and evaporated again (2-3repeats) to afford the free amine form of NB146. The analytically pureproduct was obtained by passing the above product through a short columnof Amberlite CG50 (NH₄ ⁺ form). The column was first washed with amixture of MeOH/H₂O (3:2), then the product was eluted with a mixture ofMeOH/H₂O/NH₄OH (8:1:1) to afford NB146 (0.175 grams, 68% yield). For thestorage and biological tests, compound was converted to its sulfate saltform: the free base was dissolved in water, the pH was adjusted around7.0 with H₂SO₄ (0.1 N) and lyophilized.

¹HNMR (500 MHz, CD₃OD): “Ring I”: δ_(H)=1.21 (d, 3H, J=6.0 Hz, CH₃),2.71 (dd, 1H, J₁=4.2, J₂=10.3 Hz, H-2′), 3.21 (t, 1H, J=10.0 Hz, H-4′),3.48 (t, 1H, J=9.6 Hz, H-3′), 3.81 (dd, 1H, J₁=3.4, J₂=10.0 Hz, H-5′),4.09 (m, 1H, H-6′), 5.16 (d, 1H, J=2.5 Hz, H-1′); “Ring II”: δ_(H)=1.10(m, 1H, H-2_(ax)), 2.14 (td, 1H, J₁=4.5, J₂=12.5 Hz, H-2_(eq)), 2.49 (m,1H, H-1), 2.69 (m, 1H, H-3), 3.20 (m, 2H, H-4 and H-6), 3.44 (t, 1H,J=9.1 Hz, H-5). The additional peaks in the spectrum were identified asfollows: δ_(H)=0.97 (t, 3H, J=7.2 Hz), 1.57 (m, 2H), 2.49 (m, 1H), 2.71(m, 1H).

¹³CNMR (125 MHz, CD₃OD): δ_(C)=11.9, 16.6, 23.7, 34.6 (C-2), 49.7, 51.4,57.4, 58.9, 67.8, 73.5, 75.8, 76.5, 76.6, 77.8, 90.8, 103.2 (C-1′).

MALDI TOFMS calculated for C₁₆H₃₄N₃O₇ ([M+H]⁺) m/e: 380.2; measured m/e:380.1.

Synthesis of NB147

NB147 was prepared according to Scheme 3 presented hereinbelow (reagentsand conditions: a) 5.5 equivalents Ac₂O, Py, −20° C., 24 hours; b)BF₃—OEt₂, MS, CH₂Cl₂, −30° C., 3 hours; c) THF, 0.5M NaOH, 60° C., 24 h;d) PMe3, NaOH, THF, room temperature), starting with Compound 2c (theprecursor of the NB146, see Scheme 2 hereinabove).

Briefly, Compound 2c was selectively acetylated to afford the requiredacceptor A which was then glycosylated with trichloroacetimidate donor Bas previously described (Nudelman, I. et al., Bioorg. Med. Chem. Lett.,2006, 16, pp. 6310-6315) to give the corresponding trisaccharide C at91% isolated yield. Two subsequent deprotection steps that included:treatment with strong base (NaOH, 60° C.) to remove all the ester andamide protections and Staudinger reaction to convert azides to theamines obtained the target NB147 at two steps yield of 81%. The finalproduct, along with all the intermediates were characterized by all thestandard analytical techniques including ¹H, 1³C and 2D-NMR, along with1D-TOXY to assign the structures of the products.

Synthesis of NB147

Compound 2c (750 mg, 1.0 equivalents) was dissolved in anhydrouspyridine (8 mL) and cooled to −20° C. At this temperature, aceticanhydride (2.0 mL, 5.6 equivalents) was added dropwise and allowed thereaction to progress at −20° C. The reaction progress was monitored byTLC, which indicated completion after 17 hours. The reaction mixture wasdiluted with EtOAc, and extracted with aqueous solution of NaHCO₃, HCl(2%), saturated aqueous NaHCO₃, and brine. The combined organic layerswere dried over anhydrous MgSO₄ and concentrated. The crude product waspurified by silica gel column chromatography to afford Compound A (600mg, 54% yield). Anhydrous CH₂Cl₂ (15 mL) was added to a powdered,flame-dried 4 Å molecular sieves (2.0 grams), followed by the additionacceptor A (500 mg, 1.0 equivalents) and the known donor B (2.5 grams,4.0 equivalents). The reaction mixture was stirred for 10 min at roomtemperature and was then cooled to −30° C. At this temperature,catalytic amount of BF₃.Et₂O (0.15 ml) was added and the mixture wasstirred at −30° C. and the reaction progress was monitored by TLC, whichindicated the completion after 60 minutes. The reaction mixture wasdiluted with ethyl acetate and washed with saturated NaHCO₃ and brine.The combined organic layer was dried over MgSO₄, evaporated andsubjected to column chromatography (EtOAc/Hexane) to obtain the titledcompound C (715 mg) at 91% yield. Compound C from the above step (715mg) was dissolved in minimal amount of THF and treated with 0.5Msolution of NaOH and refluxed for overnight at 60° C. After which thereaction mixture was cooled to room temperature and evaporated todryness. The crude product was purified by DOWEX-H⁺ ion exchange columnto obtain the title compound D (400 mg) in 95% yield.

¹H NMR (500 MHz, CD₃OD): “Ring I”: δ_(H)=1.25 (d, 3H, J=6.0 Hz, CH₃),3.12 (dd, 1H, J₁=3.4, J₂=10.0 Hz, H-2′), 3.34 (t, 1H, J=9.0 Hz, H-4′),3.96 (m, 1H, H-3′ and H-5′), 4.04 (m, 1H, H-6′), 6.00 (d, 1H, J=3.2 Hz,H-1′); “Ring II”: δ_(H)=1.20 (m, 1H, H-2ax), 2.27 (td, 1H, J₁=4.5,J₂=12.5 Hz, H-2eq), 2.54 (m, 1H, H-1), 3.24 (t, 1H, J=9.0 Hz, H-6), 3.50(m, 1H, H-3), 3.64 (t, 1H, J=9.5 Hz, H-5), 3.72 (t, 1H, J=9.0 Hz, H-4);“Ring III”: δ_(H)=3.48-3.59 (m, 2H, H-5″ and H-5″), 4.01 (m, 1H, H-4″),4.05 (m, 1H, H-3″) 4.15 (m, 1H, H-2″), 5.36 (s, 1H, H-1″). Theadditional peaks in the spectrum were identified as follows: δ_(H)=0.98(t, 3H, J=7.2 Hz), 1.55 (m, 2H), 2.50 (m, 1H), 2.70 (m, 1H).

¹³C NMR (125 MHz, CD₃OD): δ_(C)=11.9, 17.9, 23.8, 32.7, 49.7, 54.4,58.0, 62.3, 64.9, 69.3, 72.5, 72.6, 74.4, 75.1, 76.3 (2C), 76.9, 82.4,86.2, 97.3 (C-1′), 110.7 (C-1″).

MALDI TOFMS: calculated for C₂₁H₃₇N₁₀O₁₀ ([M+H]⁺) m/e: 589.2; measuredm/e: 589.1.

To a stirred solution of Compound D from the above step (380 mg, 1.0equivalents) in a mixture of THF (3 mL) and aqueous NaOH (1 mM, 5 mL),PMe₃ (1 M solution in THF, 5 mL, 7.8 equivalents) was added. Theprogress of the reaction was monitored by TLC [CH₂Cl₂/MeOH/H₂O/MeNH₂(33% solution in EtOH), 10:15:6:15], which indicated completion after 3hours. The reaction mixture was purified by flash chromatography on ashort column of silica gel. The column was washed with the followingsolvents: THF (100 mL), CH₂Cl₂ (100 mL), EtOH (50 mL), and MeOH (100mL). The product was then eluted with the mixture of 5% MeNH₂ solution(33% solution in EtOH) in 80% MeOH. Fractions containing the productwere combined and evaporated under vacuum. The pure product was obtainedby passing the above product through a short column of Amberlite CG50(NH₄ form). First, the column was washed with water, then the productwas eluted with a mixture of 10% NH₄OH in water to yield compound NB147(230 mg, 67% yield). For the storage and biological tests, compoundNB147 was converted to its sulfate salt form as follow. The free baseform was dissolved in water, the pH was adjusted to 6.7 with H₂SO₄ (0.1N) and lyophilized to afford the sulfate salt of NB147.

¹HNMR (500 MHz, CD₃OD): “Ring I”: δ_(H)=1.22 (d, 3H, J=6.0 Hz, CH₃),2.61 (dd, 1H, J₁=3.4, J₂=9.0 Hz, H-2′), 3.23 (t, 1H, J=10.0 Hz, H-4′),3.54 (t, 1H, J=9.6 Hz, H-3′), 3.81 (dd, 1H, J=3.4, J₂=10.0 Hz, H-5′),4.12 (m, 1H, H-6′), 5.20 (d, 1H, J=3.5 Hz, H-1′); “Ring II”: δ_(H)=1.12(m, 1H, H-2ax), 2.10 (td, 1H, J=4.5, J₂=12.5 Hz, H-2eq), 2.49 (m, 1H,H-1), 2.75 (m, 1H, H-3), 3.32 (t, 1H, J=9.0 Hz, H-6), 3.38 (t, 1H, J=9.5Hz, H-4), 3.52 (t, 1H, J=9.0 Hz, H-5); “Ring III” δ_(H): 2.80 (dd, 1H,J=7.0, J₂=13.5 Hz, H-5″), 2.94 (dd, 1H, J₁=4.3, J₂=13.5 Hz, H-5″), 3.86(m, 1H, H-4″), 3.96 (t, 1H, J=5.4 Hz, H-3″), 4.07 (m, 1H, H-2″), 5.26(d, 1H, J=2.6 Hz, H-1″). The additional peaks in the spectrum wereidentified as follows: δ_(H)=0.97 (t, 3H, J=7.2 Hz), 1.53 (m, 2H), 2.49(m, 1H), 2.71 (m, 1H).

¹³CNMR (125 MHz, CD₃OD): δ_(C)=11.9, 16.7, 23.7, 34.7 (C-2), 45.2, 49.7,52.5, 57.9, 58.6, 67.9, 72.5, 73.6, 75.3, 76.3, 76.4, 76.7, 84.9, 85.6,87.1, 102.0 (C-1′), 110.3 (C-1″).

MALDI TOFMS: calculated for C₂₁H₄₃N₄O₁₀ ([M+H]⁺) m/e: 511.2; measuredm/e: 511.1.

Synthesis of NB150 (Shown as its TFA Acid Addition Salt)

NB150 was prepared according to Scheme 4 presented hereinbelow, startingwith Compound 1. Briefly, the guanidinylation of the free N-1 amine byprotected guanidinylation reagent and Et₃N as a base afforded thedesired Compound 3. Boc deprotection was carried out by TFA to produceCompound 4 with free amines on the guanidinium moiety. Finally,Staudinger reaction was used to remove the azide protection, resultingin the final product NB150 (Scheme 4, reagents and conditions: (a) Et₃N,H₂O/Dioxane, 81% (b) TFA, CH₂Cl₂, 0° C.→25° C. (c) (i) PMe₃, THF, NaOH0.1M, (ii) The product was eluted from the ion exchange column with amixture of 2% TFA in MeOH, at 2 steps yield of 83%).

To a solution of compound 1 (2.69 grams, 1 equivalents) in H₂O (1 mL)was added 1,4-dioxane (5 mL) and N,N′-diBoc-N″-triflylguanidine (4.05grams, 1.5 equivalents) in alternating portions so the solution remainedrelatively clear. After 5 min, NEt₃ (3 mL, 3 equivalents) was added atroom temperature. After 24 hours, the 1,4-dioxane was evaporated, theremaining residue and H₂O was extracted with CHCl₃(3×10 mL), washed withH₂O and brine, and dried over MgSO₄. The guanidinylated product isolatedby flash column chromatography on silica gel (CHCl₃/MeOH) compound 3(3.51 grams, 81%).

¹H NMR (500 MHz, CDCl₃): ‘Ring I’: δ_(H)=5.37 (d, 1H, J=3.6 Hz, H-1),4.03 (t, 1H, J=9.7 Hz, H-3), 4.04-4.00 (m, 1H, H-6), 3.81 (dd, 1H,J=9.7, 5.7 Hz, H-5), 3.58 (t, 1H, J=9.3 Hz, H-4), 3.37 (dd, 1H, J=10.5,4.2 Hz, H-2), 1.31 (d, 3H, J=5.7 Hz, CH3-6); ‘Ring II’: δ_(H)=4.19-4.10(m, 1H, H-1), 3.67 (t, 1H, J=9.2 Hz, H-5), 3.54-3.47 (m, 1H, H-3),3.41-3.32 (m, 2H, H-4, H-6), 2.40 (dt, 1H, J=12.5, 4.1 Hz, H-2eq), 1.50(dd, 1H, J=19.7, 8.8 Hz, H-2ax); Additional peaks in the spectrum wereidentified as follow: 6H=11.46 (s, 1H, NH), 8.54 (d, 1H, J=7.0 Hz, NH),1.49 (s, 9H, Boc), 1.48 (s, 9H, Boc).

¹³C NMR (125 MHz, CDCl₃): δ_(C)=162.7, 157.2, 153.2, 98.7 (C1′), 84.0(Boc), 82.2, 80.3 (Boc), 77.2 (C5), 76.3, 74.1 (C4′), 73.6 (C5′), 72.3(C3′), 70.3 (C6′), 63.6, 59.4 (C3), 50.1 (C1), 33.0 (C2), 28.4 (Boc),28.2 (Boc), 19.3 (CH₃-6′).

MALDI TOFMS: calculated for C₂₄H₄₁N₉O₁₁ ([M+H]⁺) m/e 632.6; measured m/e632.6.

To a solution of Compound 3 (498 mg, 1 equivalents) in CH₂Cl₂ (15 mL) at10° C., TFA (6 mL) was added dropwise and after the addition thereaction mixture was allowed to attain the room temperature. Thereaction progress was monitored by TLC (CH₂Cl₂/MeOH 8:2) and indicatedof the completion of the reaction in 3 hours. The reaction mixture wasevaporated to dryness to get the crude product 4 (686 mg). The crudeproduct was subjected to the Staudinger reaction.

To a stirred solution of Compound 4 (686 mg, 1 equivalents) in a mixtureof THF (3.0 mL) and aqueous NaOH (1 mM, 5.0 mL), PMe₃ (1 M solution inTHF, 0.55 mL, 8 equivalents) was added dropwise and the mixture wasfurther stirred overnight. The completion of the reaction was indicatedby TLC (TFA/MeOH 1:49). The pure product was obtained by passing theabove mixture through a short column of Amberlite CG50 (NH4+ form). Thecolumn was washed with the following solvents: Hexane, THF, EtOAc, MeOHand CH₃CN. Then the product was eluted with a mixture of TFA/MeOH (1:49)to yield NB150. For the storage and biological tests, NB 150 wasdissolved in water and lyophilized to afford the TFA salt of NB150 (701mg, 83% for 2 steps).

¹H NMR (500 MHz, MeOD): ‘Ring I’: δ_(H)=5.41 (d, 1H, J=4.1 Hz, H-1),4.25 (qd, 1H, J=6.2, 1.8 Hz, H-6), 3.93 (dd, 1H, J=10.2, 2.2 Hz, H, 5),3.81 (dd, 1H, J=10.6, 8.9 Hz, H-4), 3.39-3.27 (m, 2H), 1.22 (d, 3H,J=6.4 Hz, CH₃-6); ‘Ring II’: δ_(H)=3.72 (t, 1H, J=9.6 Hz, H-5),3.62-3.52 (m, 2H, H-1, H-6), 3.48-3.35 (m, 2H, H-3, H-4), 2.30 (dt, 1H,J=12.4, 4.1 Hz, H-2eq), 1.71 (dd, 1H, J=24.9, 12.3 Hz, H-2ax).

¹³C NMR (125 MHz, MeOD): δ_(C)=159.2, 100.1 (C1′), 85.6 (C5), 77.0(C5′), 76.5, 76.3 (C4), 72.2, 71.8 (C4′), 66.0 (C6′), 56.4, 52.9, 51.0(C3), 32.1 (C2), 15.7 (CH₃-6′).

MALDI TOFMS: calculated for C₁₄H₂₉N₅O₇ ([M+H]⁺) m/e 380.4; measured m/e380.8.

NB151 and NB152 were prepared by glycosylation reactions between twodifferent acceptors 6 and 7 with donor 5, which contains guanidiniumgroup, as depicted in Scheme 5 hereinbelow, followed by deprotectionsteps.

The synthesis of acceptors 6 and 7 in Scheme 5 was performed accordingto previously published procedures (Nudelman, I. et al., Bioorg. Med.Chem., 2010, 18, pp. 3735-3746). The synthesis of donor 5 was done fromthe known ribose derivative A (reported previously in Nudelman, I. etal., Bioorg. Med. Chem. Lett., 2006, 16, pp. 6310-6315) as illustratedin the Scheme 6 hereinbelow.

Compound A was converted to Compound B by using two chemical steps inone pot reaction: reduction of azide to amine by H₂, Pd/C; the reactionof the resulted amine with guanidinium reagent and Et₃N as a base toreceive the desired Compound B. The next two steps were deprotection ofSTol with N-Bromosuccinimide (NBS) gaining Compound C and substitutionof trichloroacetimidate group for gaining the final active donor 5(Scheme 6, reagents and conditions: (a) H₂, Pd/C, DIPEA, 95% (b) NBS,Acetone/H₂O, −25° C., 83% (c) CCl₃CN, K₂CO₃, 0° C.→25° C., 50% Donor 5).

Donor 5 was prepared by stirring a solution of Compound A (6.87 grams, 1equivalents) in EtOAc (15 mL) N,N′-diBoc-N″-triflylguanidine (5.48grams, 1 equivalents; following Santana, A. G. et al., J. Org. Chem.,2010, 75(15), pp. 5371-5374), 20 mole % Pd/C (5% w/w), anddiethylisopropylamine (DIPEA) (2.71 grams, 1.5 equivalents) were added.Three vacuum/hydrogen cycles were performed, and the mixture was furtherstirred under a H₂ atmosphere (balloon) overnight. The completion of thereaction was indicated by TLC (EtOAc/Hexane 1:4). The reaction mixturewas then filtered over a Celite®pad, which was washed twice with ethylacetate, and the combined filtrates were evaporated. Columnchromatography of the residue (EtOAc/Hexane 15:85) afforded the requiredguanidinylated Compound B (9.44 grams, 95% yield).

¹H NMR (400 MHz, CDCl₃): δ_(H)=11.48 (s, 1H, NH), 8.73 (t, 1H, J=4.4 Hz,NH), 7.91 (dd, 4H J=10.0, 8.8 Hz, STol), 7.51 (t, 4H, J=8.6 Hz, Bz),7.35 (dd, 4H, J=7.9 Hz, Bz), 7.17 (d, 2H, J=7.8 Hz, Bz), 5.65 (t, 1H,J=4.5 Hz, H-2), 5.52 (d, 1H, J=4.0 Hz, H-1), 5.38 (t, 1H, J=5.4 Hz,H-3), 4.48 (dt, 1H, J=7.3, 5.3 Hz, H-4), 3.90 (ddd, 1H, J=13.6, 5.5, 4.5Hz, H-5), 3.55-3.44 (m, 1H, H-5′), 1.49 (s, 9H, Boc), 1.45 (s, 9H, Boc).

¹³C NMR (125 MHz, CDCl₃): δC=21.3 (STol), 43.1 (C-5), 72.9 (C-3), 75.1(C-2), 79.4, 80.2 (C-4), 83.3, 88.9 (C-1), 127.7, 128.5 (2C), 129.2(2C), 129.9, 130.1, 133.5 (2C), 134.7, 139.1, 153.1 (Boc), 156.5 (Boc),163.5 (Boc), 165.1 (Bz), 165.3 (Bz).

MALDI TOFMS: calculated for C₃₇H₄₃N₃O₉S ([M+H]⁺) m/e 706.8; measured m/e706.6.

A stirred solution of Compound B (3 grams, 1 equivalent) in a mixture ofAcetone/H₂O (50:5 mL) was cooled to −25° C. After stirring for 10 min,NBS (3 grams, 4 equivalents) was added in portions. The progress of thereaction was monitored by TLC (EtOAc/Hexane 1:4) and indicated that thereaction was completed in 1.5 hours. At this stage the reaction mass wasdiluted with EtOAc (50 mL). The diluted solution was extracted withNaHCO₃ (2×30 mL). Then the organic layer was washed with saturate NaClsolution and dried over anhydrous MgSO₄. The solvent was evaporated todryness and subjected column chromatography (EtOAc/Hexane 1:4) to yieldCompound C (13.3 grams, 83%).

MALDI TOFMS: calculated for C₃₀H₃₇N₃O₁₀ ([M+H]⁺) m/e 600.6; measured m/e600.9.

A stirred solution of Compound C (6.66 grams, 1 equivalents) indistilled CH₂Cl₂ (85 mL) under argon atmosphere was cooled to 0° C.After stirring for 10 minutes, CCl₃CN (12.82 grams, 8 equivalents) wasadded dropwise. Then K₂CO₃ (4.6 grams, 3 equivalents) and dried MgSO₄(8.5 grams) were added. After stirring for 30 minutes at 0° C., themixture was allowed to warm to room temperature and stirred overnight.The completion of the reaction was indicated by TLC (EtOAc/Hexane 1:4).The reaction mixture was then filtered over a Celite®pad, which waswashed twice with EtOAc, and the combined filtrates were evaporated.Column chromatography of the residue (EtOAc/Hexane 15:85+1 ml Et₃N)afforded the required donor 5 (4 grams, 48%).

¹H NMR (500 MHz, CDCl₃): δ_(H)=11.41 (s, 1H, NH), 8.67 (s, 1H, NH), 8.64(t, 1H, J=5.4 Hz, NH), 7.96 (dd, 2H, J=8.2, 1.1 Hz, Bz), 7.90 (dd, 2H,J=8.2, 1.0 Hz, Bz), 7.56 (t, 1H, J=7.5 Hz, Bz), 7.51 (t, 1H, J=7.5 Hz,Bz), 7.40 (t, 2H, J=7.9 Hz, Bz), 7.33 (t, 2H, J=7.9 Hz, Bz), 6.54 (s,1H, H-1), 5.91 (d, 1H, J=4.8 Hz, H-2), 5.68 (dd, 1H, J=7.0, 4.9 Hz,H-3), 4.71 (td, 1H, J=7.2, 4.7 Hz, H-4), 4.03 (ddd, 1H, J=14.0, 6.3, 4.9Hz, H-5), 3.79 (ddd, 1H, J=13.9, 7.3, 4.9 Hz, H-5′), 1.43 (s, 9H, Boc),1.41 (s, 9H, Boc).

¹³C NMR (125 MHz, CDCl₃): δ_(C)=28.1 (Boc), 28.3 (Boc), 43.5 (C5), 72.6(C3), 74.9 (C2), 80.9 (C4), 102.7 (C1), 128.5, 128.6, 129.9, 130.0,133.5, 133.7, 153.0, 156.6, 160.6, 163.5, 165.0, 165.4.

MALDI TOFMS: calculated for C₃₂H₃₇C₁₃N₄O₁₀ ([M+H]⁺) m/e 745.0; measuredm/e 745.5.

Synthesis of NB151 (Shown as its TFA Acid Addition Salt)

NB151 was prepared starting with the acceptor 6, and donor 5 asillustrated in Scheme 7 hereinbelow (Reagents and conditions: (a)BF₃.Et₂O, CH₂Cl₂, −30° C., 54% (b) MeNH₂, 52% (c) TFA, CH₂Cl₂, 0° C.→25°C. (d) (i) PMe₃, THF, NaOH 0.1M, (ii) The product was eluted from theion exchange column with a mixture of 2% TFA in MeOH, 85% for 2 steps).

Anhydrous CH₂Cl₂ (19 mL) was added to a powdered flame dried 4 Åmolecular sieves (1.6 grams), followed by the addition of acceptor 6(142 mg, 1 equivalents) and donor 5 (546 mg, 3 equivalents). The mixturewas cooled down to −50° C. and BF₃-Et₂O was added dropwise. The progressof the reaction was monitored by TLC (EtOAc/Hexane 3:7), and indicatedthat the reaction was completed in 30 minutes. The reaction was dilutedwith EtOAc, and filtered through a pad of Celite®. After thoroughwashing of the Celite® with EtOAc, the washes were combined andevaporated to dryness. The crude was purified by flash chromatography(EtOAc/Hexane 3:7) to yield Compound A (496 mg, 40%).

¹H NMR (500 MHz, CDCl₃): ‘Ring I’: δ_(H)=5.92 (d, 1H, J=4.0 Hz, H-1),5.38 (dd, 1H, J=10.7, 9.3 Hz, H-3), 4.98 (dd, 1H, J=6.7, 2.0 Hz, H-6),4.95 (dd, 1H, J=10.5, 9.3 Hz, H-4), 4.45 (dd, 1H, J=10.6, 2.0 Hz, H-5),3.80 (dd, 1H, J=10.9, 3.9 Hz, H-2), 1.24 (d, 3H, J=6.7 Hz, CH₃-6); ‘RingII’: δ_(H)=5.02 (t, 1H, J=9.9 Hz, H-6), 3.83 (t, 1H, J=9.4 Hz, H-5),3.74 (t, 1H, J=9.7 Hz, H-4), 3.58-3.46 (m, 2H, H-1, H-3), 2.38 (dt, 1H,J=13.2, 4.7 Hz, H-2eq), 1.48 (dd, 1H, J=26.5, 12.9 Hz, H-2ax); ‘RingIII’: δ_(H)=5.62 (d, 1H, J=4.3 Hz, H-1), 5.57 (s, 1H, H-3), 5.30 (dd,1H, J=7.3, 5.2 Hz, H-2), 4.58 (dt, 1H, J=7.4, 2.7 Hz, H-4), 4.06 (ddd,1H, J=14.5, 6.0, 3.9 Hz, H-5), 3.59 (ddd, 1H, J=13.8, 8.4, 3.8 Hz, H-5);Additional peaks in the spectrum were identified as follow: δ_(H)=11.53(s, 1H, NH), 8.72 (dd, 1H, J=6.2, 4.2 Hz, NH), 7.92-7.87 (m, 4H, Bz),7.57-7.49 (m, 2H, Bz), 7.39-7.32 (m, 4H, Bz), 2.07 (s, 3H, Ac), 2.05 (s,3H, Ac), 2.04 (s, 3H, Ac), 1.69 (s, 3H, Ac), 1.54 (s, 9H, Boc), 1.46 (s,9H, Boc).

¹³C NMR (125 MHz, CDCl₃): δ_(C)=170.2 (Ac), 170.2 (Ac), 170.1 (Ac),169.9 (Ac), 165.6 (Bz), 165.2 (Bz), 163.5, 156.4, 153.4, 133.8 (Bz),133.6 (Bz), 129.9 (Bz), 129.8 (Bz), 128.6 (Bz), 128.5 (Bz), 108.1 (C3″),96.5 (C1′), 80.1 (C5), 79.5 (C4″), 77.5 (C4), 74.7 (C1″), 73.7 (C6),72.2 (C2″), 71.1 (C3′), 70.2 (C5′), 69.2 (C4′), 68.7 (C6′), 61.6 (C2′),58.9, 58.6, 43.8 (C5″), 32.4 (C2), 28.3 (Boc), 28.3 (Boc), 21.3 (Ac),21.0 (Ac), 20.9 (Ac), 20.7 (Ac), 13.7 (CH₃-6′).

MALDI TOFMS: calculated for C₅₁H₆₄N₁₂O₂₀ ([M+H]+) m/e 1166.1; measuredm/e 1166.1.

Compound A (495 mg) was dissolved in a solution of MeNH₂ (33% solutionin EtOH, 20 mL) at room temperature overnight. The completion of thereaction was indicated by TLC (MeOH/EtOAc 1:4). Thereafter, the reactionmixture was evaporated to dryness. The crude product was subjected tocolumn chromatography (MeOH/EtOAc 1:4) to yield Compound B (175 mg,52%).

¹H NMR (500 MHz, MeOD): ‘Ring I’: δ_(H)=5.95 (d, 1H, J=3.0 Hz, H-1),4.03-3.99 (m, 1H, H-5), 3.98-3.90 (m, 2H, H-3, H-6), 3.38 (t, 1H, J=8.9Hz, H-4), 3.17 (dd, 1H, J=10.6, 5.2 Hz, H-2), 1.24 (d, 3H, J=4.5 Hz,CH₃-6); ‘Ring II’: δ_(H)=3.69 (t, 1H, J=10.0 Hz, H-4), 3.62 (t, 1H,J=9.6 Hz, H-5), 3.54 (ddd, 1H, J=15.4, 10.9, 4.4 Hz, H-1), 3.48-3.40 (m,1H, H-3), 3.37 (t, J=9.9 Hz, H-6), 2.21 (dt, 1H, J=11.7, 4.0 Hz, H-2eq),1.32 (dd, 1H, J=26.2, 13.1 Hz, H-2ax); ‘Ring III’: δ_(H)=5.35 (s, 1H,H-1), 4.21 (d, 1H, J=4.3 Hz, H-2), 4.06-3.99 (m, 1H, H-3), 3.85 (dd, 1H,J=14.2, 1.1 Hz, H-4), 3.43 (dd, 1H, J=13.9, 1.3 Hz, H-5); Additionalpeaks in the spectrum were identified as follow: δ_(H)=1.55 (s, 9H,Boc), 1.49 (s, 9H, Boc).

¹³C NMR (125 MHz, MeOD): δ_(C)=164.4, 157.7, 154.0, 111.4 (C1″), 97.6(C1′), 85.2 (C5), 81.6, 77.6, 77.0 (C4), 76.4 (C2″), 75.0, 74.4, 73.0,72.4, 69.7, 64.6, 62.1, 61.5, 45.0 (C5″), 33.3 (C2), 28.6 (Boc), 28.4(Boc), 18.3 (CH₃-6′).

MALDI TOFMS: calculated for C₂₉H₄₈N₁₂O₁₄ ([M+Na]⁺) m/e 811.7; measuredm/e 811.8.

To a solution of Compound B (175 mg, 1 equivalents) in CH₂Cl₂ (10 mL) at−10° C., TFA (3.2 mL) was added dropwise and after the addition thereaction mixture was allowed to attain the room temperature. Thereaction progress was monitored by TLC (CH₂Cl₂/MeOH 8:2) and indicatedof the completion of the reaction in 3 hours. The reaction mixture wasevaporated to dryness to get the crude product C (185 mg). The crudeproduct was subjected to the Staudinger reaction.

To a stirred solution of Compound C (185 mg, 1 equivalents) in a mixtureof THF (3 mL) and aqueous NaOH (1 mM, 5.0 mL), PMe₃ (1 M solution inTHF, 3 mL, 7.8 equivalents) was added dropwise and the mixture wasfurther stirred overnight. The completion of the reaction was indicatedby TLC (TFA/MeOH 1:49). The pure product was obtained by passing theabove mixture through a short column of Amberlite CG50 (NH₄+ form). Thecolumn was washed with the following solvents: Hexane, THF, EtOAc, MeOHand CH₃CN. Then the product was eluted with a mixture of TFA/MeOH (1:49)to yield NB151. For the storage and biological tests, NB151 wasdissolved in water and lyophilized to afford the TFA salt of NB151 (574mg, 85% for 2 steps).

¹H NMR (500 MHz, MeOD): ‘Ring I’: δ_(H)=5.54 (d, 1H, J=4.0 Hz, H-1),4.30-4.20 (m, 1H, H-6), 3.90 (dd, 1H, J=9.6, 1.9 Hz, H-5), 3.83 (t, 1H,J=9.6 Hz, H-3), 3.40-3.29 (m, 2H, H-2, H-4), 1.20 (d, 3H, J=7.3 Hz,CH₃-6); ‘Ring II’: δ_(H)=3.97 (t, 1H, J=9.8 Hz, H-4), 3.82 (t, 1H, J=8.9Hz, H-5), 3.64 (t, 1H, J=9.7 Hz, H-6), 3.51 (ddd, 1H, J=18.4, 14.2, 8.3Hz, H-1), 3.31-3.21 (m, 1H, H-3), 2.48 (dt, 1H, J=12.6, 3.9 Hz, H-2eq),1.85 (dd, 1H, J=25.1, 12.3 Hz, H-2ax); ‘Ring III’: δ_(H)=5.31 (d, 1H,J=4.0 Hz, H-1), 4.09 (t, 1H, J=4.9 Hz, H-2), 4.06-3.97 (m, 2H, H-3,H-4), 3.56-3.46 (m, 2H, H-5).

¹³C NMR (125 MHz, MeOD): δ_(C)=159.1, 110.9 (C1″), 98.5 (C1′), 85.1,82.5, 82.0 (C4), 77.5 (C5′), 75.6 (C2″), 73.6 (C6), 71.8, 71.7, 71.3,66.2 (C6′), 55.8, 50.9 (C1), 50.9 (C3), 44.5 (C5″), 29.6 (C2), 15.85(CH₃—C6′).

MALDI TOFMS: calculated for C₁₉H₃₈N₆O₁₀ ([M+H]⁺) m/e 511.5; measured m/e511.9.

Synthesis of NB152 (Shown as its TFA Acid Additional Salt)

NB152 was prepared starting with the acceptor 7 and Donor 5 asillustrated in Scheme 7 (reagents and conditions: (a) BF₃-Et₂O, CH₂Cl₂,−30° C., 40% (b) MeNH₂, 78% (c) TFA, CH₂Cl₂, 0° C.-25° C. (d) (i) PMe₃,THF, NaOH 0.1M, (ii) The product was eluted from the ion exchange columnwith a mixture of 2% TFA in MeOH, 88% for 2 steps).

To a powdered, flame dried 4 Å molecular sieves (5.85 grams) was addedanhydrous CH₂Cl₂ (78 mL), followed by the addition of acceptor 7 (755mg, 1 equivalents) and donor 5 (2.3 grams, 3 equivalents). The mixturewas cooled down to −50° C. and BF₃-Et₂O was added dropwise. The progressof the reaction was monitored by TLC (EtOAc/Hexane 3:7), and indicatedthat the reaction was completed in 10 minutes. The reaction was dilutedwith EtOAc, and filtered through a pad of Celite®. After thoroughwashing of the Celite® with EtOAc, the washes were combined andevaporated to dryness. The crude was purified by flash chromatography(EtOAc/Hexane 3:7) to yield Compound A (496 mg, 40%).

¹H NMR (500 MHz, CDCl₃): ‘Ring I’: δ_(H)=5.90 (d, 1H, J=3.9 Hz, H-1),5.39 (t, J=10.4 Hz, 1H), 4.98 (dd, 1H, J=12.2, 8.0 Hz, H-4), 4.48 (d,1H, J=10.6 Hz, H-5), 3.79 (dd, 1H, J=10.7, 3.9 Hz, H-2), 1.24 (d, 3H,J=6.7 Hz, CH₃-6); ‘Ring II’: δ_(H)=4.91 (t, 1H, J=10.1 Hz, H-6),4.03-3.96 (m, 1H, H-1), 3.93 (t, 1H, J=9.2 Hz, H-5), 3.71 (t, 1H, J=9.4Hz, H-4), 3.64-3.54 (m, 1H, H-3), 2.54 (dt, 1H, J=12.6, 4.1 Hz, H-2eq),1.35 (dd, 1H, J=24.8, 12.3 Hz, H-2ax); ‘Ring III’: δ_(H)=5.69 (d, 1H,J=4.0 Hz, H-1), 5.61 (s, 1H, H-3), 5.37 (dd, 1H, J=7.0, 5.4 Hz, H-2),4.57 (dd, 1H, J=8.3, 4.8 Hz, H-4), 4.11-4.01 (m, 1H, H-5), 3.61-3.50 (m,1H, H-5); Additional peaks in the spectrum were identified as follow:δ_(H)=11.54 (s, 1H), 8.72 (t, J=4.3 Hz, 1H), 7.99-7.78 (m, 6H, Bz),7.42-7.30 (m, 4H, Bz), 5.18 (dd, 1H, J=6.7, 5.0 Hz), 3.54-3.45 (m, 2H),2.14-2.02 (m, 1H), 1.57-1.52 (m, 1H), 2.29 (s, 3H, Ac), 2.21 (s, 3H,Ac), 2.07 (s, 3H, Ac), 2.06 (s, 3H, Ac), 2.05 (s, 3H, Ac), 1.54 (s, 9H,Boc), 1.45 (s, 9H, Boc).

¹³C NMR (125 MHz, CDCl₃): δ_(C)=177.1, 170.2 (Ac), 170.1 (Ac), 170.0(Ac), 169.9 (Ac), 165.5 (Bz), 165.1 (Bz), 164.1, 157.3, 153.6, 133.7(Bz), 129.7 (Bz), 128.8 (Bz), 108.1 (C3″), 96.6 (C1′), 80.5 (C5), 78.9(C4″), 77.7 (C4), 74.8 (C1″), 73.3 (C6), 72.2, 71.5, 70.9, 70.4 (C5′),68.6 (C4′), 61.6 (C2′), 58.7 (C3), 49.0 (C1), 43.8 (C5″), 32.9 (C2),28.3 (Boc), 28.1 (Boc), 21.0 (Ac), 20.9 (Ac), 20.5 (Ac), 13.9 (CH₃-6′).

MALDI TOFMS: calculated for C₅₇H₇₃N₁₃O₂₃ ([M+H]⁺) m/e 1309.3; measuredm/e 1309.7.

Compound A (50 mg) was dissolved in a solution of MeNH₂ (33% solution inEtOH, 2 mL) at room temperature overnight. The completion of thereaction was indicated by TLC (MeOH/EtOAc 1:4). After the completion ofthe reaction, the reaction mixture was evaporated to dryness. The crudeproduct was subjected to column chromatography (MeOH/EtOAc 1:49) toyield Compound B (27 mg, 78%).

¹H NMR (500 MHz, MeOD): ‘Ring I’: δ_(H)=5.98 (d, 1H, J=2.8 Hz, H-1),4.08-3.91 (m, 2H, H-5, H-6), 3.96 (t, 1H, J=9.5 Hz, H-3), 3.36 (t, 1H,J=9.6 Hz, H-4), 3.18 (dd, 1H, J=10.8, 5.2 Hz, H-2), 1.26 (d, 3H, J=3.9Hz, CH₃-6); ‘Ring II’: δ_(H)=3.63-3.72 (m, 2H, H-1, H-4, H-5), 3.54-3.58(m, 1H, H-1), 3.34-3.38 (m, 2H, H-3, H-6), 2.15 (dt, 1H, J=12.9, 4.0 Hz,H-2eq), 1.48 (dd, 1H, J=25.0, 12.7 Hz, H-2ax); ‘Ring III’: δ_(H)=5.37(s, 1H, H-1), 4.19 (d, 1H, J=4.0 Hz, H-2), 4.01 (s, 1H, H-3), 3.88 (d,1H, J=15.2 Hz, H-4), 3.38 (d, 2H, J=14.4 Hz, H-5); Additional peaks inthe spectrum were identified as follow: δ_(H)=4.15 (dd, 1H, J=3.9, 8.8Hz), 3.53-3.44 (m, 2H), 2.13-1.99 (m, 1H), 1.92-1.82 (m, 1H), 1.54 (s,9H, Boc), 1.47 (s, 9H, Boc).

¹³C NMR (125 MHz, MeOD): δ_(C)=177.4, 164.5, 157.7 (Boc), 154.0 (Boc),111.4 (C1″), 97.6 (C1′), 86.1, 81.6, 80.5, 77.1, 76.5 (C2″), 75.7 (C5″),75.1, 74.5, 73.2, 72.3, 70.2, 69.4, 64.6 (C2′), 61.9 (C1), 50.5, 47.5,45.3, 34.7, 32.1 (C2), 28.4 (Boc), 26.3 (Boc), 18.1 (CH₃-6′).

MALDI TOFMS: calculated for C₃₃H₅₅N₁₃O₁₆ ([M+Na]⁺) m/e 912.8; measuredm/e 912.7.

To a solution of Compound B (109 mg, 1 equivalents) in CH₂Cl₂ (3.3 mL)at −10° C., TFA (1.3 mL) was added dropwise and after the addition thereaction mixture was allowed to attain the room temperature. Thereaction progress was monitored by TLC (CH₂Cl₂/MeOH 8:2) and indicatedof the completion of the reaction in 2 hours. The reaction mixture wasevaporated to dryness to get the crude product C (169 mg). The crudeproduct was subjected to the Staudinger reaction.

To a stirred solution of Compound C (169 mg, 1 equivalents) in a mixtureof THF (3 mL) and aqueous NaOH (1 mM, 5.0 mL), PMe₃ (1 M solution inTHF, 2.74 mL, 7.8 equivalents) was added dropwise and the mixture wasfurther stirred overnight. The completion of the reaction was indicatedby TLC (TFA/MeOH 1:49). The pure product was obtained by passing theabove mixture through a short column of Amberlite CG50 (NH₄ ⁺ form). Thecolumn was washed with the following solvents: Hexane, THF, EtOAc, MeOHand CH₃CN. Then the product was eluted with a mixture of TFA/MeOH (1:49)to yield NB152. For the storage and biological tests, NB152 wasdissolved in water and lyophilized to afford the TFA salt of NB152 (350mg, 88% for 2 steps).

¹H NMR (500 MHz, MeOD): ‘Ring I’: δ_(H)=5.52 (d, 1H, J=3.9 Hz, H-1),4.22 (d, 1H, J=5.8 Hz, H-6), 3.88 (d, 1H, J=9.1 Hz, H-5), 3.81 (t, 1H,J=9.6 Hz, H-4), 3.36-3.28 (m, 2H, H-2, H-3), 1.19 (d, 3H, J=6.32 Hz,CH₃-6); ‘Ring II’: δ_(H)=3.93-3.83 (m, 2H, H-1, H-4), 3.75 (t, 1H, J=9.1Hz, H-5), 3.60 (t, 1H, J=9.7 Hz, H-6), 3.45-3.37 (m, 1H, H-3), 2.20 (dt,1H, J=13.1, 3.8 Hz, H-2eq), 1.69 (dd, 1H, J=25.2, 12.4 Hz, H-2ax); ‘RingIII’: δ_(H)=5.28 (d, 1H, J=3.5 Hz, H-1), 4.07 (t, 1H, J=4.3 Hz, H-2),4.04-3.96 (m, 2H, H-3, H-4), 3.49 (t, 2H, J=5.2 Hz, H-5); Additionalpeaks in the spectrum were identified as follow: δ_(H)=4.21 (dd, 1H,J=4.5, 8.9 Hz), 3.14-3.01 (m, 2H), 2.15-2.03 (m, 1H), 2.03-1.97 (m, 1H).

¹³C NMR (125 MHz, MeOD): δ_(C)=176.2, 159.2, 111.2 (C1″), 98.5 (C1′),86.0 (C5), 82.44, 82.1, 77.4, 75.8 (C2″), 74.7 (C6), 71.8, 71.8, 71.4(C4′), 71.0 (C6′), 66.2, 55.9, 51.4 (C3), 49.8, 44.5 (C5″), 37.8, 32.7,31.7 (C2), 15.9 (CH₃-6′).

MALDI TOFMS: calculated for C₂₃H₄₅N₇O₁₁ ([M+H]⁺) m/e 612.6; measured m/e612.9.

Example 2 Readthrough Activity in Cell-Based Assay of Compounds ofExample 1

Experimental Method:

Suppression of nonsense mutations (readthrough activity) by the testedcompounds according to embodiments of the present invention was testedin vitro using reporter plasmids harboring a mutation in the chosengene, as described, for example, in U.S. Pat. No. 8,895,519 and byVecsler, M. et al. [PLoS ONE, 2011, 6(6) p. e20733].

Briefly, HEK-293T cells were transfected by the plasmids, and 24 hourspost transfection the cells were lysed and tested for the expressionlevels of the firefly luciferase and renilla luciferase. Wild-type (WT)plasmids expressed both firefly luciferase and renilla luciferase whilemutant plasmids only expressed the renilla luciferase due to the stopcodon found in the inserted sequence. In the tested compounds'readthrough activity assays, the compounds were added to the cells'suspension 6 hours post-transfection. In case the compounds exertedsuppression of the premature nonsense/stop codon mutation, the fireflyluciferase was expressed and a fold-change in its expression wasobserved.

Results:

To determine whether the tested compounds can induce the functionalsuppression of disease-causing nonsense mutations in human cells, thesynthesis of firefly luciferase and renilla luciferase from cDNAscontaining naturally occurring premature stop codon mutations that causeRett syndrome were assayed. In all cases, the mutations introduce anin-frame ochre (UGA) stop codon in place of arginine residue, R168X,R270X and R294X mutations, which result in UGAG, UGAA and UGAUtetranucleotide termination signals, respectively.

Readthrough activity of Rett syndrome mutations was tested using thecompounds presented in Table 1, and the mutation suppression wascalculated based on firefly/renilla ratio values, normalized the valuewith the same ratio obtained without a tested compound (control), andcompare the result to the expression levels observed in the WT. Ingeneral, since the renilla reporter gene is situated upstream withrespect to the tested gene, and the firefly reporter gene is situateddownstream, readthrough activity can be quantified by calculating theratio of downstream expression to upstream expression (firefly/renillaexpression ratio) and noting the proportion (percent) of this ratio withrespect to the same measurements using the WT sequence, namely asnormalized fractions of the expression level ratio observed for the WT.Alternatively, the firefly/renilla expression ratio can be normalizedwith respect to the firefly/renilla expression ratio observed in thecontrol experiment (no readthrough-inducing compound). Since thefirefly/renilla expression ratio in the WT is essentially insensitive tothe presence of the readthrough-inducing compound, and the controlexperiment is essentially also insensitive to the presence of thereadthrough-inducing compound, as none is present, the two normalizationmethods are expected to show similar trends, as seen in the resultspresented hereinbelow.

Measuring the same firefly/renilla expression ratios using the samecompounds and control, but using the WT sequence, will signify theeffect of the tested compounds on general expression level, regardlessof the readthrough activity, thereby indicating if the tested compoundexerts protein synthesis inhibition activity, as typical aminoglycosideantibiotics do. The WT measurements are also indicative of theexperimental error.

Hence, if a given readthrough-inducing compound, according to someembodiments of the present invention, exerts some readthrough activity,the measurements will show a large firefly/renilla expression ratiocompared to the firefly/renilla expression ratio observed for thecontrol (no readthrough-inducing compound), and a high proportionalvalue (in the order of hundreds percent). If there is no readthroughactivity, the firefly/renilla expression ratios for both the inactivecompound and the control are expected to be small absolutely and similarproportionally, giving a value of about 100%.

FIGS. 2A-C present comparative bar plot showing readthrough levels ofthe Rett syndrome causing premature stop codon mutations R168X (FIG.2A), R270X (FIG. 2B) and R294X (FIG. 2C), as measured and calculated forthe compounds presented in Table 1 being contacted with expression cellsat a concentration of 0.3 mM and 1 mM, as well as for a control sample(no added compound), based on the firefly/renilla expression ratiosversus the expression ratios observed in the WT.

FIGS. 3A-C present comparative bar plot showing readthrough levels ofthe Rett syndrome causing premature stop codon mutations R168X (FIG.3A), R270X (FIG. 3B) and R294X (FIG. 3C), as measured and calculated forthe compounds presented in Table 1 being contacted with expression cellsat a concentration of 0.3 mM and 1 mM, as well as for a control sample(no added compound), and presented as fractions of the firefly/renillaexpression ratios observed for the control sample (100%) and compared tothe expression ratios observed in the WT.

As can be seen in FIGS. 2A-C, the exemplary compounds according to someembodiments of the present invention exhibited a notable anddose-dependent readthrough activity in all three Rett syndrome mutationmodels. Compounds NB150 and NB151 presented similar readthrough activityto the level shown for the aminoglycoside antibiotic agent G418(Geneticin) at 0.3 and 1 mM doses. This result may be associated withthe significant cytotoxicity of the G418 that in turn was associatedwith an overall limited readthrough level.

As can be seen in FIGS. 3A-C, the readthrough activity compared withcontrol (non-treated cells) is unaffected in the wild type cells(approx. 100%); however, in all three Rett syndrome mutation modelsthere is a significant and dose-dependent impact of the differenttreatments on the readthrough activity (>100%). Compounds NB150, NB151and NB152 presented similar readthrough activity to the level shown forthe aminoglycoside antibiotic agent G418 (Geneticin) at 0.3 and 1 mMdoses. This result may be associated with the significant cytotoxicityof the G418 that in turn was associated with an overall limitedreadthrough level.

Example 3 Readthrough Activity in Cell-Free Assay of Compounds ofExample 1

Experimental Method:

The plasmids were transcribed in vitro and translated using rabbitreticulocytes (TNT mix) and then tested for the expression levels of thefirefly and renilla luciferases.

WT plasmids expressed both firefly and renilla luciferases while mutantplasmids expressed only the renilla luciferase due to the stop codonfound in the inserted sequence. The readthrough assays were conductedfor the tested compounds and the controls by adding the compounds to thein vitro transcription/translation reaction mixture. In case thecompounds exerted suppression of the premature nonsense/stop codonmutation, the firefly luciferase was expressed and a fold-change in itsexpression was observed.

Results:

Readthrough activity of Cystic Fibrosis (CF) mutation G542X was testedusing the compounds presented in Table 1, and the mutation suppressionwas calculated based on firefly/renilla expression ratio values, andnormalized with respect to the expression level of the WT and thecontrol sample (no tested compound).

FIGS. 4A-F present the results of cystic fibrosis G542X nonsensemutation suppression dose-response cell-free assays conducted for threeexemplary compounds according to embodiments of the present invention,NB144, NB145 and NB146, at a concentration rage of 0-50 aM, wherein FIG.4A shows the expression level of the firefly luciferase which is founddownstream of the WT sequence, FIG. 4B shows the expression level of thefirefly luciferase which is found downstream of the G542X mutantsequence, FIG. 4C shows the expression level of the renilla luciferasewhich is found upstream of the WT sequence, FIG. 4D shows the expressionlevel of the renilla luciferase which is found upstream of the G542Xmutant sequence, FIG. 4E shows the firefly/renilla expression ratiomeasured in the WT sequence, and FIG. 4F shows the firefly/renillaexpression ratio measured in the G542X mutant sequence.

As can be seen in FIGS. 4A-F, the expression levels up or downstream ofthe WT sequence are grossly insensitive to the concentration of thetested compounds, with a relatively small decrease the expression levelsat high concentrations of the tested compound, presumably due to theresidual protein synthesis inhibitory effect thereof (see, FIGS. 4A, 4Cand 4E). In sharp contrast, the expression levels downstream of themutant sequence showed an intense dose-dependent response, which is notseen upstream of the mutant sequence (see, FIGS. 4B and 4D), thereforethe downstream to upstream expression level ratio (firefly/renillaexpression ratio) also exhibits an intense dose-dependent response,indicative of the mutation suppression activity of the tested compounds(FIG. 4F).

FIGS. 5A-B present the results of cystic fibrosis G542X nonsensemutation suppression dose-response cell-free assays conducted for threeexemplary compounds according to embodiments of the present invention,NB144, NB145 and NB146, at a concentration rage of 0-50 μM, wherein FIG.5A shows the expression level of the firefly luciferase, which is founddownstream of the mutant sequence, as a fraction of the expression levelexhibited in the control experiment (no added compound), and FIG. 5Bshows the firefly/renilla expression ratio, down and upstream of themutant sequence, as a fraction of the expression level in the controlexperiment.

As can be seen in FIGS. 5A-B, the mutation suppression activity of thethree exemplary compounds, according to embodiments of the presentinvention, is clearly dose-dependent for all three compounds, andparticularly for NB146, which also shown more protein synthesisinhibitory effect (see, FIGS. 4A, 4C and 4E), particularly for thefirefly luciferase gene.

FIGS. 6A-F present the results of cystic fibrosis G542X nonsensemutation suppression dose-response cell-free assays conducted for threeexemplary compounds according to embodiments of the present invention,NB150, NB151 and NB152, at a concentration rage of 0-50 aM, wherein FIG.6A shows the expression level of the firefly luciferase which is founddownstream of the WT sequence, FIG. 6B shows the expression level of thefirefly luciferase which is found downstream of the G542X mutantsequence, FIG. 6C shows the expression level of the renilla luciferasewhich is found upstream of the WT sequence, FIG. 6D shows the expressionlevel of the renilla luciferase which is found upstream of the G542Xmutant sequence, FIG. 6E shows the firefly/renilla expression ratiomeasured in the WT sequence, and FIG. 6F shows the firefly/renillaexpression ratio measured in the G542X mutant sequence.

FIGS. 7A-B present the results of cystic fibrosis G542X nonsensemutation suppression dose-response cell-free assays conducted for threeexemplary compounds according to embodiments of the present invention,NB150, NB151 and NB152, at a concentration rage of 0-50 μM, wherein FIG.7A shows the expression level of the firefly luciferase, which is founddownstream of the mutant sequence, as a fraction of the expression levelexhibited in the control experiment (no added compound), and FIG. 7Bshows the firefly/renilla expression ratio, down and upstream of themutant sequence, as a fraction of the expression level in the controlexperiment.

As can be seen in FIGS. 6A-F and FIGS. 7A-B, also compounds NB150, NB151and NB152 exhibit essentially the same mutation suppression activity asobserved for the exemplary compounds NB144, NB145 and NB146 in FIGS.4A-F and FIGS. 5A-B, namely a notable dose-dependent readthroughactivity, which is correlated to some extent to protein synthesisinhibition, as seen for NB152, particularly for the renilla luciferasegene.

FIGS. 8A-C present the results of Rett syndrome R168X (FIG. 8A), R270X(FIG. 8B) and R294X (FIG. 8C) nonsense mutations suppression cell-freeassays conducted for six exemplary compounds according to embodiments ofthe present invention, NB144, NB145, NB146, NB150, NB151 and NB152, at aconcentration of 5 μM. As shown therein, when compared with the control(non-treated cell extracts), the wild type cell extracts are unaffected(approx. 100%); however, in all three Rett syndrome mutation modelsthere is a significant impact of the different treatments on thereadthrough activity (>>>100%).

As can be seen in FIGS. 8A-C, the readthrough activity of the testedcompounds is notably more substantial than the protein synthesisinhibitory effect, demonstrating the effectiveness of the testedexemplary compounds in suppressing the nonsense mutations whileexhibiting a relatively low degree of the inhibitory side effect. Amongthe N1-substituted derivatives, NB146 exhibited a better activitycompared to NB144 and NB145; and among the guanidine derivatives thepseudo-trisaccharide NB152 showed a higher activity compared to NB150and NB151. These data suggest that inclusion of a hydrophobic moiety atthe N1 position has a pronounced effect on the biological effect ofaminoglycosides.

Example 4 Chemical Syntheses of Exemplary Diol-ContainingAminoglycosides According to Some Embodiments of the Present Invention

General Techniques:

NMR spectra (including 1H, 13C, DEPT, 2D-COSY, 1D TOCSY, HMQC, HMBC)were routinely recorded on a Bruker Avance™ 500 spectrometer, andchemical shifts reported (in ppm) are relative to internal Me₄Si (δ=0.0)with CDCl₃ as the solvent, and to MeOD (δ=3.35) as the solvent. ¹³C NMRspectra were recorded on a Bruker Avance™ 500 spectrometer at 125.8 MHz,and the chemical shifts reported (in ppm) relative to the solvent signalfor CDCl₃ (δ=77.00), or to the solvent signal for MeOD (δ=49.0).

Mass spectra analyses were obtained either on a Bruker Daltonix Apex 3mass spectrometer under electron spray ionization (ESI) or by a TSQ-70Bmass spectrometer (Finnigan Mat).

Reactions were monitored by TLC on Silica Gel 60 F254 (0.25 mm, Merck),and spots were visualized by charring with a yellow solution containing(NH₄)Mo₇O₂₄.4H₂O (120 grams) and (NH₄)₂Ce(NO₃)₆ (5 grams) in 10% H₂SO₄(800 mL).

Flash column chromatography was performed on Silica Gel 60 (70-230mesh).

All reactions were carried out under an argon atmosphere with anhydroussolvents, unless otherwise indicated.

G418 (geneticin) and gentamicin were purchased from Sigma. All otherchemicals and biochemicals, unless otherwise indicated, were obtainedfrom commercial sources.

Compounds NB153, NB 155, NB156 and NB157, presented in Table 3 below,are prepared essentially as described hereinabove and in further detailhereinbelow.

All the structures were confirmed by a combination of various 1D and 2DNMR techniques, including 1D TOCSY, 2D COSY, 2D ¹H-¹³C HMQC and HMBC,along with mass spectrometry.

TABLE 3 Compound Structure NB153

NB155

NB156

NB157

Syntheses of Pseudo-Disaccharides NB153 and NB155:

NB153 and NB155 pseudo-disaccharides are two diastereomers at C6′position of the 6′,7′-diol, exhibiting 6′-(R) configuration and 6′-(S)configuration, respectively.

The syntheses of compounds NB153 and NB155 are illustrated in Scheme 8below.

Briefly, the perazido derivative 18 was selectively protected by TIPSCl,and the remaining hydroxyls were protected by pmethoxybenzyl (PMB)groups to afford 19. Selective deprotection of silyl group with TBAF wasfollowed by oxidation with 2-iodoxy benzoic acid (IBX) and Wittigreaction to afford the terminal alkene 20. The alkene 20 wasdihydroxylated to provide the diol 21 as an inseparable mixture of6-diastereomers. Treatment of 21 with DDQ was followed by acetylation(Ac₂O) and deacetylation (NaOMe) steps to afford the mixture of6′-diastereomers (about 3:1 ratio), which was successfully separated bycolumn chromatography to give the major diastereomer 22 and the minordiastereomer 23. The absolute configuration at 6′-position was assignedby using ¹H-NMR magnetic anisotropy, as detailed hereinbelow, whichestablished 6-(R)- and 6-(S)-configuration for the major and minordiastereomers, respectively. The two diastereomers 22 and 23 wereseparately subjected to Staudinger reaction to produce thepseudodisaccharides NB153 and NB155, respectively.

Synthesis of (2R, 3S, 4R, 5R, 6S)-5-azido-6-(((1R, 2R, 3S, 4R, 6S)-4,6-diazido-2,3-dihydroxycyclohexyl)oxy)-2-(hydroxymethyl)tetrahydro-2H-pyran-3,4-diol(Compound 18)

Compound 18 was prepared according to previously published procedure[Nyffeler et al. J. Am. Chem. Soc. 2002, 124, 10773-10778]. Briefly, theparomamine (1.0 gram, 3.0 mmol), NaHCO₃ (3.1 grams, 36.9 mmol) andcopper (II) sulfate (6 mg, 0.24 mmol) were dissolved in water (5.0 mL).Triflic azide stock solution prepared from Tf₂O (4.6 mL, 27.6 mmol) andNaN₃ (3.6 grams, 55.7 mmol) was added followed by the addition ofmethanol (40 mL) to reach the homogeneous solution. The reaction mixture(blue color) was stirred vigorously at room temperature and thecompletion of the reaction was monitored by the change of blue color togreen. After stirring for 48 hours, TLC (EtOAc/MeOH 95:5) analysisindicated the completion of the reaction. The solvents were evaporatedto dryness and the residue was subjected to column chromatography (EtOAc100%) to thereby obtain compound 18 (650 mg, 52% yield).

¹H NMR (500 MHz, MeOD): ‘Ring I’: δ=H 5.69 (d, 1H, J=3.7 Hz, H-1), 3.99(ddd, 1H, J=9.9, 4.1, 2.6 Hz, H-5), 3.94 (dd, 1H, J=10.2, 9.1 Hz, H-3),3.84 (dd, 1H, J=11.9, 2.3 Hz, H-6), 3.78 (dd, 1H, J=11.8, 4.4 Hz, H-6),3.46 (dd, 1H, J=9.7, 9.3 Hz, H-4), 3.13 (dd, 1H, J=10.5, 3.7 Hz, H-2);‘Ring II’: 6H 3.80 (t, 1H, J=8.8 Hz, H-5), 3.77-3.67 (m, 3H, H-1, H-3,H-4), 3.56 (t, 1H, J=9.6 Hz, H-6), 2.59-2.48 (m, 1H), 1.68 (dd, 1H,J=26.3, 12.7 Hz, H-2).

¹³C NMR (125 MHz, MeOD): δ=C 99.3 (C1′), 80.7, 77.8 (C5), 77.7 (C6),73.9 (C5′), 72.4 (C3′), 71.6, 64.8 (C2′), 62.1 (C6′), 61.6, 60.9, 33.1(C2).

MALDI TOFMS: calculated for C₁₂H₁₉N₉O₇ ([M+K]+) m/e 440.3; measured m/e440.2.

Synthesis of (((2R, 3S, 4R, 5R, 6S)-5-azido-6-(((1R, 2R, 3S, 4R,6S)-4,6-diazido-2,3-bis((4-methoxybenzyl)oxy)cyclohexyl)oxy)-3,4-bis((4-methoxybenzyl)oxy)tetrahydro-2H-pyran-2-yl)methoxy)triisopropylsilane (Compound 19)

Compound 18 (11.6 grams, 28.9 mmol) was dissolved in anhydrous DMF (80mL) and cooled to 0° C. Triisopropylsilyl chloride (TIPSCl, 8 mL, 37.3mmol) was added dropwise, followed by addition of 4-DMAP (10.6 grams,86.7 mmol). The reaction mixture was allowed to attain the roomtemperature under stirring, and the reaction progress was monitored byTLC (EtOAc/Hexane 7:3), which indicated the completion after 5 hours.The reaction mixture was diluted with ethyl acetate (50 mL) and H₂O (20mL), and the two layers were separated. The aqueous layer was thoroughlywashed with ethyl acetate (4×30 mL). The combined organic layers werewashed with sat. NaCl solution and dried over anhydrous MgSO₄. Thesolvent was evaporated to dryness and the residue was subjected tocolumn chromatography (EtOAc/Hexane 25:75) to yield the correspondingsilyl ether (18a) (13.3 grams, 83%).

¹H NMR (500 MHz, CDCl₃): ‘Ring I’: δ=H 5.14 (d, 1H, J=4.0 Hz, H-1),4.09-4.02 (m, 2H, H-3, H-6), 3.98 (td, 1H, J1=8.0, J2=4.5 Hz, H-5), 3.82(dd, 1H, J1=9.5, J2=8.0 Hz, H-6), 3.66 (t, 1H, J=9.0 Hz, H-4), 3.48 (dd,1H, J1=10.5, J2=4.0 Hz, H-2); ‘Ring II’: δ=H 3.52 (t, 1H, J=8.0 Hz,H-5), 3.47-3.37 (m, 2H, H-1, H-6), 3.34-3.22 (m, 2H, H-3, H-4), 2.29(dt, 1H, J1=12.0, J2=4.0 Hz, H-2eq), 1.47 (ddd, 1H, J1=J2=J3=12.0 Hz,H-2ax); The additional peaks in the spectrum were identified as follow:δ=H 1.16-1.09 (m, 3H, TIPS), 1.07 (s, 12H, TIPS), 1.06 (s, 6H, TIPS).

¹³C NMR (125 MHz, CDCl₃): δ=C 99.3 (C1′), 83.4 (C4), 76.1 (C5), 75.5(C6), 75.1 (C4′), 72.6 (C3′), 69.6 (C5′), 66.0 (C6′), 63.5 (C2′), 59.8(C1), 58.9 (C3), 32.1 (C2), 17.9 (2C, TIPS), 11.8 (TIPS).

MALDI TOFMS: calculated for C₂₁H₃₉N₉O₇Si ([M+Na]+) m/e 580.6; measuredm/e 580.3.

To a stirred solution of the silyl ether from above (9.82 grams, 17.6mmol) and sodium hydride (3.38 grams, 140 mmol) in DMF (200 mL), wasadded p-Methoxybenzyl chloride (14.3 mL, 105.3 mmol) at 0° C. Thereaction progress was monitored by TLC (EtOAc/Hexane 3:7). After 8 hoursthe reaction was completed and ice was added in small portions to quenchthe reaction. The mixture was diluted with ethyl acetate (100 mL) andwashed with water (2×50 mL). The combined aqueous layers were extractedwith diethyl ether (2×50 mL); the combined organic layers were driedover anhydrous MgSO₄, and evaporated to dryness. The residue waspurified by column chromatography (EtOAc/Hexane 8:92) to thereby obtaincompound 19 (15.28 grams, 84%).

¹H NMR (500 MHz, CDCl₃): ‘Ring I’: δ=H 5.45 (d, 1H, J=3.5 Hz, H-1), 3.94(m, 2H, H-3, H-5), 3.88-3.78 (m, 2H, H-6), 3.59 (t, 1H, J=9.5 Hz, H-4),3.17 (dd, 1H, J1=10.5, J2=3.5 Hz, H-2); ‘Ring II’: δ=H 3.56-3.42 (m, 2H,H-4, H-5), 3.41-3.32 (m, 1H, H-1), 3.32-3.20 (m, 2H, H-3, H-6), 2.17(dt, 1H, J1=12.5, J2=4.0 Hz, H-2eq), 1.34 (ddd, 1H, J1=J2=J3=12.5 Hz,H-2ax); The additional peaks in the spectrum were identified as follow:δ=H 7.21 (d, 2H, J=8.0 Hz, PMB), 7.17 (d, 6H, J=8.0 Hz, PMB), 6.85-6.72(m, 8H, PMB), 4.86 (d, 1H, J=10.0 Hz, PMB), 4.80-4.65 (m, 6H, PMB), 4.61(d, 1H, J=10.0 Hz, PMB), 3.74-3.68 (m, 12H, PMB), 1.04-0.94 (m, 21H,TIPS).

¹³C NMR (125 MHz, CDCl₃): δ=C 159.5 (PMB), 159.4 (PMB), 159.3 (PMB),159.2 (PMB), 130.7 (PMB), 130.3 (PMB), 130.2 (PMB), 129.9 (PMB), 129.8(PMB), 129.7 (PMB), 129.3 (PMB), 128.7 (PMB), 113.9 (2C, PMB), 97.5(C1′), 84.5, 84.4, 79.8, 77.9 (C4′), 76.9, 75.6 (PMB), 75.2 (PMB), 74.9(PMB), 74.5 (PMB), 72.9, 63.5 (C2′), 62.3 (C6′), 60.3 (C1), 59.5, 55.3(4C, PMB), 32.4 (C2), 18.1 (2C, TIPS), 12.1 (TIPS).

MALDI TOFMS: calculated for C₅₃H₇₁N₉O₁₁Si ([M+Na]+) m/e 1061.2; measuredm/e 1061.6.

Synthesis of (2R, 3R, 4R, 5R,6R)-3-azido-2-(((1R,2R,3S,4R,6S)-4,6-diazido-2,3-bis((4-methoxybenzyl)oxy)cyclohexyl)oxy)-4,5-bis((4-methoxybenzyl)oxy)-6-vinyltetrahydro-2H-pyran (Compound 20)

To a stirred solution of compound 19 (19.82 grams, 19 mmol) in THF (230mL) at 0° C., TBAF (11.05 mL, 38.1 mmol) was added and the reactionprogress was monitored by TLC (EtOAc/Hexane 2:3). After 19 hours, thesolvent was evaporated to dryness and the obtained residue was subjectedto column chromatography (EtOAc/Hexane 3:7) to thereby obtain thecorresponding 6′-alcohol (14.74 grams, 88%).

¹H NMR (500 MHz, CDCl₃): ‘Ring I’: δ=H 5.51 (d, 1H, J=4.0 Hz, H-1), 3.98(dt, 1H, J1=8.0, J2=2.0 Hz, H-5), 3.92 (t, 1H, J=10.0 Hz, H-3), 3.70(dd, 1H, J1=12.0, J2=2.0 Hz, H-6), 3.64 (dd, 1H, J1=12.0, J2=2.0 Hz,H-6), 3.49 (dd, 1H, J1=10.0, J2=8.0 Hz, H-4), 3.17 (dd, 1H, J1=10.0,J2=4.0 Hz, H-2); ‘Ring II’: δ=H 3.53-3.44 (m, 2H, H-4, H-5), 3.38 (ddd,1H, J1=12.5, J2=10.0, J3=4.5 Hz, H-1), 3.34-3.24 (m, 2H, H-3, H-6), 2.20(dt, 1H, J1=12.5, J2=4.5 Hz, H-2eq), 1.36 (ddd, 1H, J1=J2=J3=12.5 Hz,H-2ax); The additional peaks in the spectrum were identified as follow:δ=H 7.23 (d, 2H, J=8.0 Hz, PMB), 7.20-7.14 (m, 6H, PMB), 6.83-6.75 (m,8H, PMB), 4.89 (d, 1H, J=10.0 Hz, PMB), 4.80-4.68 (m, 6H, PMB), 4.55 (d,1H, J=10.0 Hz, PMB), 3.73-3.7 (m, 12H, PMB).

¹³C NMR (125 MHz, CDCl₃): δ=C 159.5 (PMB), 159.4 (2C, PMB), 159.2 (PMB),130.2 (PMB), 130.1 (2C, PMB), 129.9 (PMB), 129.8 (PMB), 129.6 (2C, PMB),128.8 (PMB), 114.0 (2C, PMB), 113.9 (2C, PMB), 97.6 (C1′), 84.4, 84.3,79.8 (C3′), 77.4, 75.6 (PMB), 75.2 (PMB), 75.1 (PMB), 74.6 (PMB), 72.0(C5′), 63.3 (C2′), 61.4 (C6′), 60.3 (C1), 59.5, 55.3 (3C, PMB), 32.4(C2).

MALDI TOFMS: calculated for C₄₄H₅₁N₉O₁₁ ([M+Na]+) m/e 903.3; measuredm/e 903.9.

To a solution of the 6′-alcohol from the above reaction (100 mg, 0.11mmol) in ethyl acetate (5 mL), IBX (95 mg, 0.33 mmol) was added in oneportion. The resulting suspension was heated at 80° C. and stirredvigorously. After the reaction was completed (3.5 hours) as indicated byTLC (EtOAc/Hexane 2:3), the reaction was cooled to room temperature andfiltered through Celite®. The Celite® was thoroughly washed with ethylacetate (2×50 mL) and the combined organic layers were evaporated underreduced pressure. The crude product was subjected to flash columnchromatography (EtOAc/Hexane 35:65) to thereby obtain the 6′-aldehyde(85 mg, 85%).

¹H NMR (500 MHz, CDCl₃): ‘Ring I’: δ=H 9.53 (s, 1H, H-6), 5.56 (d, 1H,J=4.0 Hz, H-1), 4.60 (d, 1H, J=10.0 Hz, H-4), 3.98 (dd, 1H, J1=J2=10.0Hz, H-3), 3.52-3.45 (m, 1H, H-5), 3.17 (dd, 1H, J1=10.0, J2=4.0 Hz,H-2); ‘Ring II’: δ=H 3.53-3.43 (m, 2H, H-4, H-5), 3.37 (ddd, 1H,J1=12.0, J2=10.0, J3=4.0 Hz, H-1), 3.33-3.24 (m, 2H, H-3, H-6), 2.20(dt, 1H, J1=12.5, J2=4.0 Hz, H-2eq), 1.35 (ddd, 1H, J1=J2=J3=12.5 Hz,H-2ax); The additional peaks in the spectrum were identified as follow:δ=H 7.23 (d, 2H, J=8.0 Hz, PMB), 7.19-7.10 (m, 6H, PMB), 6.83-6.72 (m,8H, PMB), 4.89 (d, 1H, J=10.0 Hz, PMB), 4.80-4.64 (m, 6H, PMB), 4.51 (d,1H, J=10.0 Hz, PMB), 3.73 (s, 3H, PMB), 3.71 (s, 6H, PMB), 3.70 (s, 3H,PMB).

¹³C NMR (125 MHz, CDCl₃): δ=C 197.3 (CHO), 159.7 (PMB), 159.6 (2C, PMB),159.2 (PMB), 130.2 (PMB), 130.0 (PMB), 129.9 (2C, PMB), 129.7 (PMB),129.6 (PMB), 129.3 (PMB), 128.6 (PMB), 114.1 (PMB), 114.0 (3C, PMB),97.5 (C1′), 84.3, 84.2, 79.8 (C3′), 78.0, 77.6, 75.6 (PMB), 75.5 (PMB),75.2 (C4′), 75.1 (PMB), 74.8 (PMB), 62.8 (C2′), 60.2 (C1), 59.1, 55.4(PMB), 55.3 (PMB), 32.21 (C2).

MALDI TOFMS: calculated for C₄₄H₄₉N₉O₁₁ ([M+Na]+) m/e 902.3; measuredm/e 902.3.

To a cooled suspension of Methyltriphenylphosphonium Iodide (70 mg, 0.19mmol) in anhydrous THF at 0° C., n-BuLi (1.6 M in hexane, 136 μL) wasadded drop wise and the resulting yellow solution was stirred foradditional 30 minutes at 0° C. The 6′-aldehyde from the previous step(61 mg, 0.069 mmol) in anhydrous THF (0.3 mL) was thereafter added at 0°C., and the reaction was allowed to stir for additional 1.5 hours atroom temperature. After completion of the reaction, as indicated by TLC(EtOAc/Hexane 2:3), the reaction was quenched with saturated NH₄Clsolution. The layers were separated and the aqueous layer was extractedwith ether (2×10 mL). The combined organic layers were washed withbrine, dried over anhydrous MgSO₄ and evaporated to dryness. The crudeproduct was purified by flash chromatography (EtOAc/Hexane 2.5:7.5) tothereby obtain Compound 20 (27 mg, 56%).

¹H NMR (500 MHz, CDCl₃): ‘Ring I’: δ=H 5.83-5.74 (m, 1H, H-6), 5.47 (d,1H, J=4.0 Hz, H-1), 5.37 (d, 1H, J=16.5 Hz, H-7trans), 5.21 (d, 1H,J=9.5 Hz, H-7cis), 4.49 (dd, 1H, J1=9.5, J2=7.5 Hz, H-5), 3.90 (t, 1H,J=9.5 Hz, H-3), 3.25-3.14 (m, 2H, H-2, H-4); ‘Ring II’: δ=H 3.54-3.44(m, 2H, H-4, H-5), 3.38 (ddd, 1H, J1=12.0, J2=9.5, J3=4.0 Hz, H-1),3.34-3.25 (m, 2H, H-3, H-6), 2.21 (dt, 1H, J1=12.5, J2=4.0 Hz, H-2eq),1.38 (ddd, 1H, J1=J2=J3=12.5 Hz, H-2ax); The additional peaks in thespectrum were identified as follow: δ=H 7.25-7.09 (m, 8H, PMB),6.84-6.73 (m, 8H, PMB), 4.88 (d, 1H, J=10.0 Hz, PMB), 4.77 (dd, 2H,J=10.0, 2.5 Hz, PMB), 4.74-4.66 (m, 3H, PMB), 4.59 (d, 1H, J=10.5 Hz,PMB), 4.52 (d, 1H, J=10.5 Hz, PMB), 3.73 (s, 3H, PMB), 3.72 (s, 6H,PMB), 3.71 (s, 3H, PMB).

¹³C NMR (125 MHz, CDCl3): δ=C 159.5 (PMB), 159.4 (2C, PMB), 159.2 (PMB),134.9 (C6′), 130.3 (PMB), 130.2 (2C, PMB), 129.9 (2C, PMB), 129.6 (2C,PMB), 128.7 (PMB), 118.8 (C7′), 114.0 (PMB), 113.9 (2C, PMB), 97.6(C1′), 84.4, 84.3, 82.4 (C4′), 79.4 (C3′), 77.6, 75.6 (PMB), 75.3 (PMB),75.0 (PMB), 74.6 (PMB), 72.7 (C5′), 63.4 (C2′), 60.3 (C1), 59.3, 55.4(PMB), 55.3 (PMB), 32.3 (C2).

MALDI TOFMS: calculated for C₄₅H₅₁N₉O₁₀ ([M+Na]+) m/e 900.9; measuredm/e 900.5.

Synthesis of 1-((2R, 3S, 4R, 5R, 6S)-5-azido-6-(((1R, 2R, 3S, 4R,6S)-4,6-diazido-2,3-bis((4-methoxybenzyl)oxy)cyclohexyl)oxy)-3,4-bis((4-methoxybenzyl)oxy)tetrahydro-2H-pyran-2-yl)ethane-1,2-diol (Compound 21)

To a stirred solution of Compound 20 (383 mg, 0.436 mmol) in acetone (5mL), water (1.5 mL) and t-BuOH (5 mL), K₂OsO₄.2H2O (16 mg, 0.043 mmol)and NMO (181 μL) were added sequentially. The progress of the reactionwas monitored by TLC (EtOAc/Hexane 2:3), which indicated the completionafter 24 hours. The solvent was then evaporated to dryness; the residuewas dissolved in EtOAc to which an aqueous solution of Na₂S₂O₃ wasadded. The layers were separated and the organic phase was washed withbrine, dried over MgSO₄ and evaporated. The crude product was subjectedto column chromatography (EtOAc/Hexane 1:1) to thereby obtain compound21 (370 mg, 93%) as a 6′-diasteromeric mixture.

Synthesis of (2R, 3S, 4R, 5R, 6S)-5-azido-6-(((1R, 2R, 3S, 4R,6S)-4,6-diazido-2,3-dihydroxycyclohexyl)oxy)-2-((R)-1,2-dihydroxyethyl)tetrahydro-2H-pyran-3,4-diol(Compound 22) and (2R, 3S,4R, 5R, 6S)-5-azido-6-(((1R, 2R, 3S,4R,6S)-4,6-diazido-2,3-dihydroxycyclohexyl)oxy)-2-((S)-1,2-dihydroxyethyl)tetrahydro-2H-pyran-3,4-diol (Compound 23)

Compound 21 (220 mg, 1.0 equiv.) from above was stirred with2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) (383 mg, 6 equiv.) inmethylene chloride and water (20:1 v/v, 15 mL) at room temperature.After the addition of DDQ, a dark green color charge transfer complexformed immediately and slowly faded to orange color as the reactionprogressed. TLC (EtOAc/MeOH 98:2) showed that the reaction completedafter 15 hours. The solvents were then evaporated and the residue wasloaded onto the silica gel column without prior work up. Due to highpolarity of the titled compound, this column chromatography allowed theremoval of only parts of the DDQ reaction byproducts. Therefore, inorder to obtain the analytically pure product, the fractions containingthe product were combined, evaporated and the residue was then subjectedto peracetylation and deacetylation steps, as follows. The crudematerial from above was dissolved in anhydrous pyridine (5 mL) andcooled to 0° C. Acetic anhydride (0.73 mL, 9 equiv.) was added dropwise,followed by the addition of 4-DMAP (0.621 gram, 6 equiv.). Aftercompletion of the reaction (2 hours), as indicated by TLC (EtOAc/Hexane2:3), the reaction was diluted with EtOAc (20 mL) and washed with 5% HClsolution, Sat. NaHCO₃, and brine, and dried over anhydrous MgSO₄. Thesolvent was evaporated to dryness and the residue was subjected to acolumn chromatography (EtOAc/Hexane 3:7) to thereby obtain thecorresponding peracetate as an inseparable mixture of 6′-diastereomers(150 mg, 91% for 2 steps).

The peracetate (215 mg, 0.314 mmol) from above was dissolved inanhydrous MeOH (5 mL) and NaOMe (152 mg, 2.81 mmol) was added in oneportion to the stirred solution at room temperature. The reactionprogress was monitored by TLC (EtOAc/MeOH 95:5), which indicatedcompletion after 4 hours. The reaction mixture was passed through ashort silica gel column and the product was eluted with MeOH. Thefractions with the compound were combined, evaporated and the crudeproduct was subjected to an additional column chromatography (EtOAc/MeOH99:1), which allowed complete separation of the two diastereomers, themajor (Rf=0.36) and minor (Rf=0.2). The major diastereomer was laterassigned, as detailed hereinunder, as the 6′-(R)-diastereomer (Compound22) and the minor one as the 6′-(S)-diastereomer (Compound 23).

Major Diastereomer (22): ¹H NMR (500 MHz, MeOD): ‘Ring I’: δ=H 5.68 (d,1H, J=4.0 Hz, H-1), 4.04 (dd, 1H, J1=9.5, J2=4.0 Hz, H-4), 3.97-3.92 (m,1H, H-6), 3.93 (t, 1H, J=10.0 Hz, H-3), 3.79 (dd, 1H, J=11.5, 3.5 Hz,H-7), 3.70 (dd, 1H, J1=11.5, J2=7.0 Hz, H-7), 3.58 (t, 1H, J=9.5 Hz,H-5), 3.13 (dd, 1H, J1=10.0, J2=4.0 Hz, H-2); ‘Ring II’: δ=H 3.57-3.47(m, 3H, H-3, H-4, H-5), 3.44 (ddd, 1H, J1=16.5, J2=8.5, J3=4.0 Hz, H-1),3.29 (t, 1H, J=9.5 Hz, H-6), 2.26 (dt, 1H, J1=12.5, J2=4.0 Hz, H-2eq),1.43 (ddd, 1H, J1=J2=J3=12.5 Hz, H-2ax).

¹³C NMR (125 MHz, MeOD): δ=C 99.1 (C1′), 80.5, 77.9 (C6), 77.9, 74.8(C6′), 73.7 (C4′), 72.9 (C5′), 72.3 (C3′), 64.5 (C2′), 64.3 (C7′), 61.7(C1), 61.0, 33.3 (C2).

MALDI TOFMS: calculated for C₁₃H₂₇N₃O₈ ([M+H]+) m/e 432.3; measured m/e432.8.

Minor Diastereomer (23): ¹H NMR (500 MHz, MeOD): Ring I′: δ=H 5.72 (d,1H, J=3.6 Hz, H-1), 4.00 (ddd, 1H, J1=6.8, J2=6.0, J3=1.1 Hz, H-6),3.97-3.91 (m, 2H, H-5, H-3), 3.74-3.67 (m, 2H, H-7, H-7), 3.64-3.59 (m,1H, H-4), 3.10 (dd, 1H, J1=10.5, J2=3.7 Hz, H-1); ‘Ring II’: δ=H3.57-3.50 (m, 2H, H-1, H-6), 3.45-3.37 (m, 2H, H-3, H-4), 3.26 (t, 1H,J=9.5 Hz, H-5), 2.24 (dt, 1H, J1=12.8, J2=4.4 Hz, H-2eq), 1.40 (ddd, 1H,J1=J2=J3=12.5 Hz, H-2ax).

¹³C NMR (125 MHz, MeOD): δ=C 99.1 (C-1′), 80.1 (C-4), 78.0 (C-6), 77.8(C-5), 73.1 (C-5′), 72.3 (C-3′), 71.3 (C-4′), 70.8 (C-6′), 65.3 (C-7′),64.4 (C-2′), 61.7 (C-3), 61.1 (C-1), 33.3 (C-2).

MALDI TOFMS: calculated for C₁₃H₂₇N₃O₈ ([M+H]+) m/e 432.3; measured m/e432.8.

Synthesis of (2R, 3S, 4R, 5R, 6S)-5-amino-6-(((1R, 2R, 3S, 4R,6S)-4,6-diamino-2,3-dihydroxycyclohexyl)oxy)-2-((R)-1,2-dihydroxyethyl)tetrahydro-2H-pyran-3,4-diol[NB153 ((R)-diasteromer)]

To a stirred solution of compound 22 (82 mg, 0.19 mmol) in a mixture ofTHF (3 mL) and aqueous NaOH (1 mM, 5 mL), PMe₃ (1 M solution in THF,0.15 mL, 2.5 mmol) was added. The progress of the reaction was monitoredby TLC [CH₂Cl₂/MeOH/H₂O/MeNH₂ (33% solution in EtOH), 10:15:6:15], whichindicated completion after 1 hour. The reaction mixture was thereafterpurified by flash chromatography on a short column of silica gel. Thecolumn was washed with the following solvents: THF (100 mL), CH₂Cl₂ (100mL), EtOH (50 mL), and MeOH (100 mL). The product was then eluted with amixture of 5% MeNH₂ solution (33% solution in EtOH) in 80% MeOH.Fractions containing the product were combined and evaporated undervacuum. The pure product was obtained by passing the above productthrough a short column of Amberlite CG50 (NH₄ ⁺ form). First, the columnwas washed with water, then the product was eluted with a mixture of 10%NH₄OH in water, to thereby obtain NB153 (49.0 mg, 73%). For storage andbiological tests, NB153 was converted to its sulfate salt form asfollow: The free base form was dissolved in water, the pH was adjustedto 6.7 with H₂SO₄ (0.1 N) and lyophilized to afford the sulfate salt ofNB153 as white foamy solid.

¹H-NMR (500 MHz, MeOD, —NH2 form): ‘Ring I’: δ=H 5.18 (d, 1H, J=4.0 Hz,H-1), 3.98-3.93 (m, 1H, H-6), 3.90 (dd, 1H, J1=10.0, J2=4.0 Hz, H-4),3.76 (dd, 1H, J1=11.5, J2=4.0 Hz, H-7), 3.70 (dd, 1H, J1=11.5, J2=6.0Hz, H-7), 3.51 (t, 1H, J=10.0 Hz, H-3), 3.44 (m, 1H, H-5), 2.74 (dd, 1H,J1=10.0, J2=4.0 Hz, H-2); ‘Ring II’: δ=H 3.43 (t, 1H, J=9.0 Hz, H-5),3.20 (t, 1H, J=9.0 Hz, H-4), 3.10 (t, 1H, J=9.5 Hz, H-6), 2.77 (ddd, 1H,J1=10.5, J2=9.0, J3=5.0 Hz, H-3), 2.66 (ddd, 1H, J1=10.5, J2=9.5, J3=5.0Hz, H-1), 2.02 (dt, 1H, J1=12.5, J2=4.0 Hz, H-2eq), 1.22 (ddd, 1H,J1=J2=J3=12.5 Hz, H-2ax).

¹³C NMR (125 MHz, MeOD): δC 102.9 (C-1′), 90.0 (C-4), 78.2 (C-6), 77.5,75.6 (C-3′), 74.3 (C-4′), 73.6 (C-6′), 73.3, 63.3 (C-7′), 57.1 (C-2′),52.4 (C-3), 51.3 (C-1), 36.7 (C2).

MALDI TOFMS: calculated for C₁₃H₂₇N₃O₈ ([M+H]+) m/e 354.3; measured m/e354.8.

Synthesis of (2R, 3S, 4R, 5R, 6S)-5-amino-6-(((1R, 2R, 3S, 4R,6S)-4,6-diamino-2,3-dihydroxycyclohexyl)oxy)-2-((S)-1,2-dihydroxyethyl)tetrahydro-2H-pyran-3,4-diol[NB155 ((S)-diastereomer)]

To a stirred solution of Compound 23 (52 mg, 0.12 mmol) in a mixture ofTHF (3 mL) and aqueous NaOH (1 mM, 5 mL), PMe₃ (1 M solution in THF,0.15 mL, 2.5 mmol) was added. The progress of the reaction was monitoredby TLC [CH₂Cl₂/MeOH/H₂O/MeNH₂ (33% solution in EtOH), 10:15:6:15], whichindicated completion after 1 hour. The reaction mixture was purified byflash chromatography on a short column of silica gel. The column waswashed with the following solvents: THF (100 mL), CH₂Cl₂ (100 mL), EtOH(50 mL), and MeOH (100 mL). The product was then eluted with the mixtureof 5% MeNH₂ solution (33% solution in EtOH) in 80% MeOH. Fractionscontaining the product were combined and evaporated under vacuum. Thepure product was obtained by passing the above product through a shortcolumn of Amberlite CG50 (NH₄ ⁺ form). First, the column was washed withwater, then the product was eluted with a mixture of 10% NH₄OH in waterto thereby obtain NB155 (36.0 mg, 78%). For storage and biologicaltests, NB155 was converted to its sulfate salt form as follow: The freebase form was dissolved in water, the pH was adjusted to 6.7 with H₂SO₄(0.1 N) and lyophilized to afford the sulfate salt of NB155 as whitefoamy solid.

¹H NMR (500 MHz, MeOD, —NH₂ form): ‘Ring I’: δ=H 5.28 (d, 1H, J=3.8 Hz,H-1′), 3.97 (td, 1H, J1=7.1, J2=1.0 Hz, H-6′), 3.89-3.82 (m, 1H, H-4′),3.63 (d, 2H, J=7.2 Hz, H-7′, H-7′), 3.59-3.51 (m, 2H, H-5′, H-3′), 2.72(m, 1H, H-2′); ‘Ring II’: δ=H 3.41 (t, 1H, J=9.1 Hz, H-5), 3.20 (t, 1H,J=9.2 Hz, H-4), 3.08 (t, 1H, J=9.4 Hz, H-6), 2.74 (m, 1H, H-3), 2.64(ddd, 1H, J1=12.2, J2=9.7, J3=4.1 Hz, H-1), 2.00 (dt, 1H, J1=12.9,J2=4.1 Hz, H-2eq), 1.21 (ddd, 1H, J1=J2=J3=12.3 Hz, H-2ax).

¹³C NMR (125 MHz, MeOD): δ=C 102.9 (C-1′), 89.6 (C-4), 79.0 (C-6), 77.9(C-5), 75.8 (C-3′), 72.3 (C-4′), 71.1 (C-5′), 70.2 (C-6′), 63.2 (C-7′),57.1 (C-2′), 52.4 (C-1), 51.4 (C-3), 37.7 (C-2).

MALDI TOFMS: calculated for C₁₃H₂₇N₃O₈ ([M+H]+) m/e 354.3; measured m/e354.8.

Syntheses of Pseudo-Trisaccharides NB156 and NB157

The syntheses of compounds NB156 and NB157 are illustrated in Scheme 9below, and were accomplished from the intermediate Compound 22 by usingessentially the same chemical transformations as for NB153 and NB155.

Briefly, regioselective acetylation of Compound 22 with Ac₂O at lowtemperature gave the corresponding C5 acceptor Compound 24. For theglycosylation of 24 the trichloroacemidate donors 25 and 26 whichfurnished the corresponding pseudo-trisaccharides 27 and 28 in 85% and93% isolated yields, respectively, exclusively as β-anomers. Treatmentwith methylamine was followed by Staudinger reaction to afford NB156 andNB157.

Synthesis of NB156 Synthesis of (2R, 3S, 4R, 5R, 6S)-6-(((1R, 2S, 3S,4R, 6S)-3-acetoxy-4,6-diazido-2-hydroxycyclohexyl)oxy)-5-azido-2-((R)-1,2-diacetoxyethyl)tetrahydro-2H-pyran-3,4-diyldiacetate (24)

Compound 22 (370 mg, 0.857 mmol) was dissolved in anhydrous pyridine (8mL) and cooled to −20° C. Acetic anhydride (0.45 mL, 4.8 mmol) was addeddropwise and the reaction was allowed to progress at −20° C. Thereaction progress was monitored by TLC, which indicated completion after17 hours. The reaction mixture was diluted with EtOAc, and extractedwith aqueous solution of HCl (2%), saturated aqueous NaHCO₃, and brine.The combined organic layers were dried over anhydrous MgSO₄ andconcentrated. The crude product was purified by silica gel columnchromatography (EtOAc/Hexane 3:7) to afford Compound 24 (292 mg, 53%yield).

¹H NMR (500 MHz, CDCl₃): ‘Ring I’: δ=H 5.45 (dd, 1H, J1=10.5, J2=9.3 Hz,H-3′), 5.37 (d, 1H, J=3.5 Hz, H-1′), 5.19 (ddd, 1H, J1=7.6, J2=4.0,J3=2.0 Hz, H-6′), 5.07 (dd, 1H, J1=10.4, J2=9.2 Hz, H-4′), 4.40 (dd, 1H,J1=10.5, J2=1.8 Hz, H-5′), 4.31 (dd, 1H, J1=12.0, J2=4.1 Hz, H-7′),4.19-4.08 (m, 1H, H-7′), 3.63-3.56 (m, 1H, H-2′); ‘Ring II’: δ=H 4.91(dd, 1H, J1=12.8, J2=7.1 Hz, H-6) 3.66 (td, 1H, J1=9.6, J2=3.5 Hz, H-5),3.53 (ddd, 1H, J1=12.4, J2=10.1, J3=4.5 Hz, H-1), 3.45 (dd, 1H, J1=19.1,J2=9.2 Hz, H-4), 3.38-3.31 (m, 1H, H-3), 2.38 (dt, 1H, J1=13.2, J2=4.4Hz, H-2eq), 1.58 (ddd, 1H, J1=J2=J3=12.6 Hz, H-2ax). The additionalpeaks in the spectrum were identified as follow: δ=H 2.17 (s, 3H,CH₃CO), 2.08 (d, 9H, J=1.5 Hz, CH₃CO), 2.04 (s, 3H, CH₃CO).

¹³C NMR (125 MHz, CDCl₃): δ=C 170.7 (C═O), 170.6 (C═O), 170.2 (C═O),170.0 (C═O), 169.9 (C═O), 98.5 (C-1′), 82.9 (C-4), 75.1 (C-6), 74.6(C-5), 71.4 (C-3′), 70.0 (C-6′), 69.9 (C-5′), 68.9 (C-4′), 61.8 (C-7′),61.5 (C-2′), 58.2 (C-3), 58.0 (C-1), 32.0 (C-2), 20.96 (CH₃), 20.92(CH₃), 20.89 (CH₃), 20.86 (CH₃), 20.8 (CH₃), 20.7 (CH₃).

MALDI TOFMS: calculated for C₂₃H₃₁N₉O₁₃ ([M+Na]+) m/e 664.20; measuredm/e 664.20.

Synthesis of (2S, 3S, 4S, 5R)-2-(((1S, 2S, 3R, 5S,6R)-2-acetoxy-3,5-diazido-6-(((2S, 3R, 4R, 5S,6R)-4,5-diacetoxy-3-azido-6-((R)-1,2-diacetoxyethyl)tetrahydro-2H-pyran-2-yl)oxy)cyclohexyl)oxy)-5-(azidomethyl)tetrahydrofuran-3,4-diyldibenzoate (27)

Anhydrous CH₂Cl₂ (15 mL) was added to a powdered, flame-dried 4 Åmolecular sieves (2.0 grams), followed by the addition of acceptorCompound 24 (292 mg, 0.455 mmol) and donor Compound 25 (1.0 gram, 1.9mmol). The reaction mixture was stirred for 10 minutes at roomtemperature and was then cooled to −30° C. Catalytic amount of BF₃-Et₂O(50 μL) was added and the mixture was stirred at −30° C.; the reactionprogress was monitored by TLC, which indicated the completion after 60minutes. The reaction mixture was diluted with ethyl acetate and washedwith saturated NaHCO₃ and brine. The combined organic layer was driedover MgSO₄, evaporated and subjected to column chromatography(EtOAc/Hexane) to obtain the Compound 27 (393 mg, 85% yield).

¹H NMR (500 MHz, CDCl₃): ‘Ring I’: δ=H 5.87 (d, 1H, J=3.8 Hz, H-1),5.42-5.34 (m, 1H, H-3), 5.24-5.13 (m, 1H, H-6), 5.10-5.03 (m, 1H, H-4),4.54 (dd, 1H, J1=10.5, J2=2.2 Hz, H-5), 4.33 (dd, 1H, J1=12.0, J2=4.1Hz, H-7), 4.20 (dd, 1H, J1=11.9, J2=7.8 Hz, H-7), 3.50 (dd, 1H, J1=10.9,J2=3.8 Hz, H-2); ‘Ring II’: δ=H 5.01 (t, 1H, J=10.0 Hz, H-6), 3.87 (t,1H, J=9.3 Hz, H-5), 3.71 (t, 1H, J=9.5 Hz, H-4), 3.57-3.48 (m, 2H, H-1,H-3), 2.38 (dt, 1H, J1=12.9, J2=4.3 Hz, H-2eq), 1.52 (ddd, 1H,J1=J2=J3=12.7 Hz, H-2ax); ‘Ring III’: δ=H 5.61 (s, 1H, H-1), 5.57 (d,1H, J=4.7 Hz, H-2), 5.42-5.35 (m, 1H, H-3), 4.59-4.47 (m, 1H, H-4),3.63-3.55 (m, 2H, H-5, H-5). The additional peaks in the spectrum wereidentified as follow: δ=H 7.93 (d, 2H, J=7.1 Hz, Ar), 7.87 (d, 2H, J=7.1Hz, Ar), 7.54 (dt, 2H, J1=19.0, J2=7.4 Hz, Ar), 7.39 (t, 2H, J=7.8 Hz,Ar), 7.34 (t, 2H, J=7.8 Hz, Ar), 2.29 (s, 3H, CH₃), 2.08-2.04 (m, 12H,4×CH₃).

¹³C NMR (125 MHz, CDCl₃): δ=C 170.7 (C═O), 170.1 (C═O), 170.08 (C═O),170.06 (C═O), 169.9 (C═O), 165.5 (Ar), 165.2 (Ar), 133.8 (Ar), 133.7(Ar), 129.8 (Ar), 129.7 (Ar), 128.8 (Ar), 128.68 (Ar), 128.63 (Ar),128.5 (Ar), 107.7 (C-1″), 96.1 (C-1′), 80.9 (C-4″), 79.8 (C-5), 77.2(C-4), 74.5 (C-2″), 73.9 (C-6), 72.0 (C-3′), 70.7 (C-3″), 69.9 (C-6′),69.2 (C-5′), 68.8 (C-4′), 61.5 (C-7′), 61.4 (C-2′), 58.9 (C-3), 58.3(C-1), 53.1 (C-5″), 32.1 (C-2), 21.04 (CH₃), 21.03 (CH₃), 20.8 (CH₃),20.7 (CH₃), 20.6 (CH₃).

MALDI TOFMS: calculated for C₄₂H₄₆N₁₂O₁₈ ([M+Na]+) m/e 1029.31; measuredm/e 1029.29.

Synthesis of (2R, 3S, 4R, 5R, 6S)-5-amino-6-(((1R, 2R, 3S, 4R,6S)-4,6-diamino-2-(((2S, 3S, 4R,5R)-5-(aminomethyl)-3,4-dihydroxytetrahydrofuran-2-yl)oxy)-3-hydroxycyclohexyl)oxy)-2-((R)-1,2-dihydroxyethyl)tetrahydro-2H-pyran-3,4-diol(NB156)

The glycosylation product 27 (393 mg, 0.390 mmol) was treated with asolution of MeNH₂ (33% solution in EtOH, 15 mL) and the reactionprogress was monitored by TLC (EtOAc/MeOH 85:15), which indicatedcompletion after 12 hours. The reaction mixture was evaporated todryness and was subjected to column chromatography (MeOH/EtOAc 2:8) tothereby obtain the corresponding completely de-esterified perazidoderivative (183 mg) in 80% yield.

¹H NMR (500 MHz, MeOD): ‘Ring I’: δ=H 5.89 (d, 1H, J=3.8 Hz, H-1), 3.97(dd, 1H, J1=9.7, J2=4.6 Hz, H-5), 3.84 (dd, 2H, J1=12.0, J2=7.1 Hz, H-6,H-3), 3.69 (d, 1H, J=8.5 Hz, H-7), 3.60 (dd, 1H, J1=11.6, J2=6.4 Hz,H-7), 3.45 (dd, 1H, J1=10.0, J2=8.7 Hz, H-4), 3.06 (dd, 1H, J1=10.6,J2=4.4 Hz, H-2); ‘Ring II’: δ=H 3.62-3.54 (m, 2H, H-4, H-5), 3.50-3.43(m, 1H, H-3), 3.40-3.33 (m, 1H, H-1), 3.33-3.26 (m, 1H, H-6), 2.12 (dt,1H, J1=13.3, J2=4.4 Hz, H-2 eq), 1.29 (ddd, 1H, J1=J2=J3=12.4 Hz, H-2ax); ‘Ring III’: δ=H 5.28 (d, 1H, J=0.8 Hz, H-1), 4.11 (dd, 1H, J1=4.4,J2=0.8 Hz, H-2), 3.98 (dd, 1H, J1=7.4, J2=4.2 Hz, H-3), 3.94 (dd, 1H,J1=7.0, J2=3.4 Hz, H-4), 3.49 (dd, 1H, J1=13.3, J2=2.8 Hz, H-5), 3.41(dd, 1H, J1=13.1, J2=6.3 Hz, H-5).

¹³C NMR (125 MHz, MeOD): δ=C 111.2 (C-1″), 97.4 (C-1′), 85.2 (C-4), 82.3(C-5′), 77.6 (C-6), 76.8 (C-5), 76.3 (C-2″), 74.6 (C-6′), 73.3 (C-3″),73.2 (C-4′), 72.7 (C-4″), 72.5 (C-3′), 64.7 (C-2′), 64.1 (C-7′), 61.9(C-3), 61.4 (C-1), 54.5 (C-5″), 33.1 (C-2).

MALDI TOFMS: calculated for C₁₈H₂₈N₁₂O₁₁ ([M+Na]+) m/e 611.20; measuredm/e 611.19.

To a stirred solution of a perazido derivative from the above reaction(183 mg, 0.311 mmol) in a mixture of THF (3 mL) and aqueous NaOH (1 mM,5 mL), PMe₃ (1 M solution in THF, 0.22 mL, 3.0 mmol) was added. Theprogress of the reaction was monitored by TLC [CH₂Cl₂/MeOH/H₂O/MeNH₂(33% solution in EtOH), 10:15:6:15], which indicated completion after 1hour. The reaction mixture was purified by flash chromatography on ashort column of silica gel. The column was washed with the followingsolvents: THF (100 mL), CH₂Cl₂ (100 mL), EtOH (50 mL), and MeOH (100mL). The product was then eluted with the mixture of 5% MeNH₂ solution(33% solution in EtOH) in 80% MeOH. Fractions containing the productwere combined and evaporated under vacuum. The pure product was obtainedby passing the above product through a short column of Amberlite CG50(NH₄ ⁺ form). First, the column was washed with water, and then theproduct was eluted with a mixture of 10% NH₄OH in water to yieldCompound NB156 as a free base form (90.0 mg, 60%).

For storage and biological tests, NB156 was converted to its sulfatesalt form as follow: The free base form was dissolved in water, the pHwas adjusted to 6.7 with H₂SO₄ (0.1 N) and lyophilized to afford thesulfate salt of NB156 as white foamy solid.

¹H NMR (500 MHz, MeOD): ‘Ring I’: δ=H 5.18 (d, 1H, J=3.6 Hz, H-1), 3.91(dt, 1H, J1=6.3, J2=3.9 Hz, H-6), 3.85 (dd, 1H, J1=10.2, J2=2.8 Hz,H-5), 3.70 (dd, 1H, J1=11.5, J2=3.7 Hz, H-7), 3.64 (dd, 1H, J1=11.5,J2=6.4 Hz, H7), 3.50 (dd, 1H, J1=10.0, J2=9.0 Hz, H-3), 3.40 (t, 1H,J=9.5 Hz, H-4), 2.60 (dd, 1H, J=10.2, 3.3 Hz, H-2); ‘Ring II’: δ=H 3.44(t, 1H, J=9.2 Hz, H-5), 3.33 (dd, 1H, J1=11.0, J2=7.6 Hz, H-4), 3.13 (t,1H, J=9.5 Hz, H-6), 2.79-2.70 (m, 1H, H-3), 2.60 (td, 1H, J1=9.4, J2=4.4Hz, H-1), 1.93 (dt, 1H, J1=13.0, J2=4.0 Hz, H-2eq), 1.16 (ddd, 1H,J1=J2=J3=12.4 Hz, H-2ax); ‘Ring III’: δ=H 5.20 (d, 1H, J=2.7 Hz, H-1),4.04 (dd, 1H, J1=5.1, J2=2.8 Hz, H-2), 3.95-3.90 (m, 1H, H-3), 3.83 (dt,1H, J1=5.3, J2=3.4 Hz, H-4), 2.89 (dd, 1H, J1=13.2, J2=4.0 Hz, H-5),2.75 (dd, 1H, J1=13.2, J2=7.3 Hz, H-5).

¹³C NMR (125 MHz, MeOD): δ=C 110.6 (C-1″), 101.7 (C-1′), 86.8 (C-4),85.5 (C-5), 84.7 (C-4″), 78.8 (C-6), 76.2 (C-2″), 75.3 (C-3′), 74.7(C-5′), 73.8 (C-6′), 73.0 (C-4′), 72.5 (C-3″), 63.4 (C-7′), 57.5 (C-2′),52.5 (C-3), 52.3 (C-1), 45.2 (C-5″), 37.5 (C-2).

MALDI TOFMS: calculated for C₁₈H₃₆N₄O₁₁ ([M+H]+) m/e 485.24; measuredm/e 485.19.

Synthesis of NB157 Synthesis of (2S, 3S, 4S, 5R)-2-(((1S, 2S 3R, 5S,6R)-2-acetoxy-3,5-diazido-6-(((2S, 3R, 4R, 5S,6R)-4,5-diacetoxy-3-azido-6-((R)-1,2-diacetoxyethyl)tetrahydro-2H-pyran-2-yl)oxy)cyclohexyl)oxy)-5-((S)-1-azidoethyl)tetrahydrofuran-3,4-diyldibenzoate (Compound 28)

Anhydrous CH₂Cl₂ (15 mL) was added to a powdered, flame-dried 4 Åmolecular sieves (2.0 grams), followed by the addition of acceptorCompound 24 (265 mg, 0.413 mmol) and donor Compound 26 (0.895 gram, 1.65mmol). The reaction mixture was stirred for 10 minutes at roomtemperature and was then cooled to −30° C. At this temperature,catalytic amount of BF₃-Et₂O (50 μL) was added and the mixture wasstirred at −30° C. The reaction progress was monitored by TLC, whichindicated the completion after 60 minutes. The reaction mixture wasdiluted with ethyl acetate and washed with saturated NaHCO₃ and brine.The combined organic layer was dried over MgSO₄, evaporated andsubjected to column chromatography (EtOAc/Hexane) to obtain Compound 28(393 mg) in 93% yield.

¹H NMR (600 MHz, CDCl₃): ‘Ring I’: δ=H 5.88 (d, 1H, J=4.0 Hz, H-1), 3.58(dd, 1H, J1=10.7, J2=4.0 Hz, H-2), 5.36 (dd, 1H, J1=10.6, J2=9.3 Hz,H-3), 5.07 (dd, 1H, J1=10.5, J2=9.3 Hz, H-4), 4.53 (dd, 1H, J1=10.6,J2=2.2 Hz, H-5), 5.18 (ddd, 1H, J1=7.5, J2=4.1, J3=2.2 Hz, H-6), 4.33(dd, 1H, J1=12.0, J2=3.9 Hz, H-7), 4.19 (dd, 1H, J1=12.1, J2=7.6 Hz,H-7); ‘Ring II’: δ=H 5.01 (t, 1H, J=9.9 Hz, H-6), 3.84 (t, 1H, J=9.4 Hz,H-5), 3.71 (t, 1H, J=9.5 Hz, H-4), 3.52 (ddd, 2H, J1=12.5, J2=10.0,J3=4.6 Hz, H-1, H-3), 2.39 (dt, 1H, J1=5.2, J2=4.5 Hz, H-2eq), 1.52(ddd, 1H, J1=J2=J3=12.7 Hz, H-2ax); ‘Ring III’: δ=H 5.60 (t, 2H, J=2.3Hz, H-1, H-2), 5.41 (dd, 1H, J1=7.6, J2=4.9 Hz, H-3), 4.33 (t, 1H, J=7.3Hz, H-4), 3.77-3.64 (m, 1H, H-5), 1.24 (d, 3H, J=6.8 Hz, 6-CH₃). Theadditional peaks in the spectrum were identified as follow: δ=H7.92-7.89 (m, 2H, Ar), 7.89-7.85 (m, 2H, Ar), 7.60-7.50 (m, 2H, Ar),7.39 (t, 2H, J=7.8 Hz, Ar), 7.34 (t, 2H, J=7.9 Hz, Ar), 2.41-2.35 (m,3H, CH₃), 2.08 (s, 3H, CH₃), 2.07 (s, 3H, CH₃), 2.07 (s, 3H, CH₃), 2.05(s, 3H, CH₃).

¹³C NMR (151 MHz, CDCl₃): δ=C 170.7 (C═O), 170.3 (C═O), 170.07 (C═O),170.03 (C═O), 169.9 (C═O), 165.5 (Ar), 165.0 (Ar), 133.8 (Ar), 133.7(Ar), 129.8 (Ar), 129.7 (Ar), 128.8 (Ar), 128.6 (Ar), 128.58 (Ar),128.56 (Ar), 107.8 (C-1″), 96.1 (C-1′), 84.6 (C-4″), 79.7 (C-5), 77.6(C-4), 74.7 (C-2″), 73.7 (C-6), 72.0 (C-3″), 71.0 (C-3), 70.0 (C-6′),69.2 (C-4′), 68.9 (C-5′), 61.7 (C-2′), 61.5 (C-7′), 59.6 (C-5″), 58.9(C-1), 58.5 (C-3), 32.2 (C-2), 21.1 (CH₃), 21.0 (CH₃), 20.8 (CH₃), 20.79(CH₃), 20.78 (CH₃), 15.8 (C-6″, CH₃).

MALDI TOFMS: calculated for C₄₃H₄₈N₁₂O₁₈ ([M+Na]+) m/e 1043.32; measuredm/e 1043.30.

Synthesis of (2R, 3S, 4R, 5R, 6S)-5-amino-6-(((1R, 2R, 3S, 4R,6S)-4,6-diamino-2-(((2S, 3S, 4R,5R)-5-((S)-1-aminoethyl)-3,4-dihydroxytetrahydrofuran-2-yl)oxy)-3-hydroxycyclohexyl)oxy)-2-((R)-1,2-dihydroxyethyl)tetrahydro-2H-pyran-3,4-diol(NB157)

The glycosylation product Compound 28 (0.393 gram, 0.384 mmol) wastreated with a solution of MeNH₂ (33% solution in EtOH, 15 mL) and thereaction progress was monitored by TLC (EtOAc/MeOH 85:15), whichindicated completion after 12 hours. The reaction mixture was evaporatedto dryness and was subjected to column chromatography (MeOH/EtOAc 2:8)to obtain the corresponding completely de-esterified perazido derivative(230 mg) in 98% yield.

¹H NMR (600 MHz, MeOD): ‘Ring I’: δ=H 5.98 (d, 1H, J=3.8 Hz, H-1), 3.11(dd, 1H, J1=10.5, J2=3.8 Hz, H-2), 4.03 (dd, 1H, J1=9.7, J2=4.5 Hz,H-4), 3.96-3.88 (m, 2H, H-3, H-6), 3.50 (dd, 1H, J1=10.0, J2=8.8 Hz,H-5), 3.75 (dd, 1H, J1=11.2, J2=2.5 Hz, H-7), 3.66 (dd, 1H, J1=11.6,J2=6.5 Hz, H-7); ‘Ring II’: δ=H 3.69-3.64 (m, 1H, H-4), 3.60 (t, 1H,J=8.9 Hz, H-5), 3.52 (ddd, 1H, J1=12.3, J2=9.7, J3=4.4 Hz, H-3), 3.42(ddd, 1H, J1=11.9, J2=9.7, J3=4.4 Hz, H-1), 3.38-3.33 (m, 1H, H-6), 2.18(dt, 1H, J1=12.6, J2=4.4 Hz, H-2eq), 1.52-1.17 (m, 1H, H-2ax); ‘RingIII’: δ=H 5.31 (d, 1H, J=0.5 Hz, H-1), 4.17 (dd, 1H, J1=4.8, J2=0.6 Hz,H-2), 4.10 (dd, 1H, J1=7.2, J2=4.7 Hz, H-3), 3.78-3.70 (m, 1H, H-4),3.69-3.57 (m, 1H, H-5), 1.33 (d, 3H, J=6.7 Hz, 6-CH₃).

¹³C NMR (151 MHz, MeOD): δ=C 110.79 (C-1″), 97.41 (C-1′), 86.03 (C-4″),85.24 (C-5), 77.47 (C-6), 76.76 (C-4), 76.47 (C-2″), 74.60 (C-6′), 73.42(C-3), 73.31 (C-4′), 72.77 (C-3″), 72.60 (C-3′), 64.66 (C-2′), 64.13(C-7′), 61.96 (C-1), 61.51 (C-5′), 60.86 (C-5″), 33.17 (C-2), 16.06(C-6″, CH₃).

MALDI TOFMS: calculated for C₁₉H₃₀N₁₂O₁₁ ([M+Na]+) m/e 625.22; measuredm/e 625.20.

To a stirred solution of the perazido derivative from the above reaction(230 mg, 0.381 mmol) in a mixture of THF (3 mL) and aqueous NaOH (1 mM,5 mL), PMe₃ (1 M solution in THF, 0.22 mL, 3.0 mmol) was added. Theprogress of the reaction was monitored by TLC [CH₂Cl₂/MeOH/H₂O/MeNH₂(33% solution in EtOH), 10:15:6:15], which indicated completion after 1hour. The reaction mixture was purified by flash chromatography on ashort column of silica gel. The column was washed with the followingsolvents: THF (100 mL), CH₂Cl₂ (100 mL), EtOH (50 mL), and MeOH (100mL). The product was then eluted with the mixture of 5% MeNH₂ solution(33% solution in EtOH) in 80% MeOH. Fractions containing the productwere combined and evaporated under vacuum. The pure product was obtainedby passing the above product through a short column of Amberlite CG50(NH₄+ form). First, the column was washed with water, then the productwas eluted with a mixture of 10% NH₄OH in water to yield NB157 (123 mg,64%) in its free base form.

For storage and biological tests, NB157 was converted to its sulfatesalt form as follow: The free base form was dissolved in water, the pHwas adjusted to 6.7 with H₂SO₄ (0.1 N) and lyophilized to afford thesulfate salt of NB157 as a white foamy solid.

¹H NMR (600 MHz, MeOD): ‘Ring I’: δ=H 5.25 (d, 1H, J=3.6 Hz, H-1),4.00-3.94 (m, 1H, H-6), 3.90 (dd, 1H, J1=9.9, J2=3.5 Hz, H-5), 3.56-3.50(m, 1H, H-3), 3.47 (dd, 1H, J1=18.3, J2=8.8 Hz, H-4), 2.66 (dd, 1H,J1=10.3, J2=3.5 Hz, H-2), 3.76 (dd, 1H, J1=11.5, J2=3.7 Hz, H-7), 3.70(dd, 1H, J1=11.5, J2=6.4 Hz, H-7); ‘Ring II’: δ=H 3.48 (dd, 1H, J1=15.9,J2=6.7 Hz, H-5), 3.37 (dd, 1H, J1=16.5, J2=7.2 Hz, H-4), 3.18 (dd, 1H,J1=13.1, J2=5.6 Hz, H-6), 2.78 (dd, 1H, J1=9.9, J2=8.2 Hz, H-3), 2.64(dd, 1H, J1=22.9, J2=10.3 Hz, H-1), 1.96 (dt, 1H, J1=7. 8, J2=3. 7 Hz,H-2eq), 1.23 (ddd, 1H, J1=J2=J3=12.5 Hz, H-2ax); ‘Ring III’: δ=H 5.26(d, 1H, J=2.7 Hz, H-1), 4.05 (d, 1H, J=1.8 Hz, H-2), 4.01 (t, 1H, J=5.7Hz, H-3), 3.56 (t, 1H, J=6.3 Hz, H-4), 3.01-2.86 (m, 1H, H-5), 1.16 (d,3H, J=6.4 Hz, 6-CH₃).

¹³C NMR (151 MHz, MeOD): δ=C 109.78 (C-1″), 101.67 (C-1′), 88.61 (C-4″),86.80 (C-4), 84.86 (C-5), 78.70 (C-6), 76.28 (C-2″), 75.46 (C-3′), 74.72(C-5′), 73.79 (C-6′), 73.07 (C-4′), 72.30 (C-3″), 63.43 (C-7′), 57.55(C-2′), 52.53 (C-3), 52.35 (C-1), 50.68 (C-5″), 49.85 (C-4), 37.64(C-2), 19.37 (C-6″, CH₃).

MALDI TOFMS: calculated for C₁₉H₃₈N₄O₁₁ ([M+H]+) m/e 498.25; measuredm/e 499.26.

Determination of Absolute Configuration at 6′-Position of NB153 andNB155:

In order to determine the absolute stereochemistry at the side-chainC6′-alcohols in NB153 and NB155, the major C6′-diasteromer alcohol 31was synthesized, as illustrated in Scheme 10.

It was assumed that the change of protecting group on the secondaryalcohols would improve the yields and isolation of the intermediateproducts at various synthetic steps experienced in the pathway in Scheme8. The PMB protection in Scheme 8 was replaced with the benzylprotection shown in Scheme 10. Thus, the benzylation of TIPS protectedCompound 18a was followed by silyl deprotection with TBAF to provide the6′-alcohol 29 in good overall yields. Dess-Martin Periodinane (DMP)oxidation provided the corresponding aldehyde, which was treated withWittig reagent to provide the terminal alkene 30. Dihydroxylation stepwas followed by selective benzylation of the primary alcohol to affordthe desired 6′-alcohol 31 as a mixture of two 6′-diastereomers. Attemptsto separate this mixture by using column chromatography with severaldifferent solvent systems proved unsuccessful, and it was found that thesilylation of the mixture 31 with t-butyldimethylsilyl chloride(TBDMSCl) in the presence of imidazole proceeded very slow and with highselectivity of the major 6′-diastereomer. Using this advantage thesilylated product of the major diastereomer 32 could be isolated in itspure form. Treatment of 32 with TBAF produced the desired product 31,which was used for configuration assignment.

To assign the absolute stereochemistry at 6′ position in compound 31,the major diastereomer 31 was separately coupled with(R)-2-methoxy-2(1-naphthyl)propanoic acid [(R)-MαNP] 33 and [(S)-MαNP]34 of known absolute stereochemistry in presence of DCC, 4-DMAP and CSA6to afford the respective esters (R,X)-MαNP 35 and (S,X)-MαNP 36, asshown in Scheme 11.

Synthesis of ((2R, 3S, 4R, 5R, 6S)-5-azido-3, 4-bis(benzyloxy)-6-(((1R,2R, 3S, 4R,6S)-4,6-diazido-2,3-bis(benzyloxy)cyclohexyl)oxy)tetrahydro-2H-pyran-2-yl)methanol(29)

To a stirred solution of the silyl ether Compound 18a (0.2 gram, 0.358mmol) and sodium hydride (0.114 gram, 4.75 mmol) in DMF (5 mL), wasadded benzyl bromide (0.255 mL, 2.14 mmol) at 0° C. The reactionprogress was monitored by TLC (EtOAc/Hexane 3:7). After 8 hours thereaction was completed and ice was added in small portions to quench thereaction. The mixture was diluted with ethyl acetate (30 mL) and washedwith water (2×50 mL). The combined aqueous layers were extracted withdiethyl ether (2×50 mL); the combined organic layers were dried overanhydrous MgSO₄, and evaporated to dryness. The residue was purified bycolumn chromatography (EtOAc/Hexane 8:92) to yield perbenzylated silylether (0.243 gram, 74%).

¹H NMR (500 MHz, CDCl₃): ‘Ring I’: δ=H 5.46 (d, 1H, J=3.3 Hz, H-1), 3.97(dd, 1H, J1=17.7, J2=8.2 Hz, H-3, H-5), 3.90 (d, 1H, J=11.6, H-6), 3.84(d, 1H, J=11.0, H-6), 3.72-3.53 (m, 1H, H-4), 3.19 (dd, 1H, J1=10.6,J2=4.4 Hz, H-2); ‘Ring II’: δ=H 3.53 (m, 2H, H-4, H-5), 3.40 (td, 1H,J1=9.9, J2=5.3 Hz, H-1), 3.30 (ddd, 2H, J1=17.6, J2=15.1, J3=9.2 Hz,H-3, H-6), 2.21 (dd, 1H, J1=8.2, J2=4.2 Hz, H-2eq), 1.34 (dt, 1H,J1=J2=J3=12.9 Hz, H-2ax); The additional peaks in the spectrum wereidentified as follow: δ=H 7.28 (m, 20H, Bn), 4.94 (m, 2H, O(CH₂)Bn),4.80 (m, 6H, O(CH₂)Bn), 1.14-0.95 (m, 21H, TIPS).

¹³C NMR (125 MHz, CDCl₃): δC 138.54 (Bn), 138.17 (Bn), 138.03 (Bn),137.49 (Bn), 128.61 (Bn), 128.58 (Bn), 128.55 (Bn), 128.31 (Bn), 128.28(Bn), 128.14 (Bn), 127.99 (Bn), 127.78 (Bn), 127.72 (Bn), 127.10 (Bn),97.7 (C1′), 84.8, 84.62, 80.2, 77.3 76.0, 75.7, 75.2, 74.9, 72.9, 63.5(C2′), 62.3 (C6), 60.4 (C1), 59.5, 32.5 (C2), 18.2 (TIPS), 18.1 (TIPS),12.1 (TIPS).

To a stirred solution of perbenzylated silyl ether compound from theabove step (9.24 grams, 10.0 mmol) in THF (100 mL) at 0° C., TBAF (9.0mL, 31.0 mmol) was added and the reaction progress was monitored by TLC(EtOAc/Hexane 2:3). After 15 hours, the solvent was evaporated todryness and the obtained residue was subjected to column chromatography(EtOAc/Hexane 3:7) to yield the corresponding perbenzylated 6′-alcohol29 (7.0 grams, 91%).

¹H NMR (500 MHz, CDCl₃): ‘Ring I’: δ=H 5.60 (d, 1H, J=3.8 Hz, H-1), 4.11(d, 1H, J=10.0 Hz, H-5), 4.05 (t, 1H, J=9.7 Hz, H-3), 3.83 (dd, 1H,J1=12.0, J2=2.0 Hz, H-6), 3.76 (dd, 1H, J1=12.1, J2=2.9 Hz, H-6),3.69-3.57 (m, 1H, H-4), 3.28 (dd, 1H, J1=10.6, J2=4.6 Hz, H-2); ‘RingII’: δ=H 3.60-3.57 (m, 2H, H-4, H-5), 3.55-3.46 (m, 1H, H-1), 3.46-3.37(m, 2H, H-3, H-6), 2.31 (dt, 1H, J1=13.2, J2=4.5 Hz, H-2eq), 1.47 (ddd,1H, J1=J2=J3=10.6 Hz, H-2ax); The additional peaks in the spectrum wereidentified as follow: δ=H 7.52-7.28 (m, 20H, Bn), 5.04 (d, 1H, J=10.8Hz, O(CH₂)Bn), 4.93 (dd, 2H, J1=10.7, J2=6.0 Hz, O(CH₂)Bn), 4.90-4.86(m, 3H, O(CH₂)Bn), 4.84 (d, 1H, J=10.5 Hz, O(CH₂)Bn), 4.71 (d, 1H,J=11.2 Hz, O(CH₂)Bn).

¹³C NMR (125 MHz, CDCl₃): δ=C 138.0 (Bn), 138.0 (Bn), 137.8 (Bn), 137.3(Bn), 128.6 (Bn), 128.6 (Bn), 128.5 (Bn), 128.2 (Bn), 128.1 (Bn), 128.1(Bn), 128.0 (Bn), 128.0 (Bn), 127.7 (Bn), 127.1 (Bn), 97.7 (C1′), 84.7,84.5, 80.1 (C3′), 77.6, 77.5, 76.0, 75.6, 75.3, 75.0, 72.0 (C5′), 63.4(C2′), 61.4 (C6′), 60.3, 59.4, 32.4 (C2).

Synthesis of (2R, 3R, 4R, 5R, 6R)-3-azido-4, 5-bis(benzyloxy)-2-(((1R,2R, 3S, 4R,6S)-4,6-diazido-2,3-bis(benzyloxy)cyclohexyl)oxy)-6-vinyltetrahydro-2H-pyran(30)

To a solution of the 6′-alcohol 29 (1.0 gram, 1.31 mmol) in ethylacetate (40 mL), IBX (1.1 gram, 3.92 mmol) was added in one portion. Theresulting suspension was heated at 80° C. and stirred vigorously. Afterthe reaction was completed (3.5 hours) as indicated by TLC (EtOAc/Hexane2:3), the reaction was cooled to room temperature and filtered throughCelite®. The Celite® was thoroughly washed with ethyl acetate (2×50 mL)and the combined organic layers were evaporated under reduced pressure.The crude product was subjected to flash column chromatography(EtOAc/Hexane 35:65) to yield the 6′-aldehyde (0.925 gram, 92%).

¹H NMR (500 MHz, CDCl₃): ‘Ring I’: δ=H 9.62 (s, 1H, H-6(CHO)), 5.62 (s,1H, H-1), 4.69 (d, 1H, J=9.9 Hz, H-4), 4.01 (t, 1H, J=9.3 Hz, H-3), 3.56(dd, 1H, J1=18.0, J2=9.1 Hz, H-5), 3.19 (d, 1H, J=14.0, H-2); ‘Ring II’:δ=H 3.56 (dd, 2H, J1=18.0, J2=9.1 Hz, H-4, H-5), 3.44 (d, 1H, J=11.7 Hz,H-1), 3.37 (t, 2H, J=8.2 Hz, H-3, H-6), 2.28 (d, 1H, J1=10.2 Hz, H-2eq),1.44 (ddd, 1H, J1=J2=J3=14.0 Hz, H-2ax); The additional peaks in thespectrum were identified as follow: δ=H 7.27 (m, 20H, Bn), 5.00 (d, 1H,J=10.9 Hz, O(CH₂)Bn), 4.92-4.75 (m, 6H, O(CH₂)Bn), 4.63 (d, 1H, J=10.7Hz, O(CH₂)Bn).

¹³C NMR (125 MHz, CDCl₃): δ=C 197.2 (CHO), 138.0 (Bn), 137.5 (Bn), 137.3(Bn), 137.1 (Bn), 128.7 (Bn), 128.6 (Bn), 128.6 (Bn), 128.6 (Bn), 128.3(Bn), 128.3 (Bn), 128.2 (Bn), 128.1 (Bn), 97.6 (C1′), 84.6, 84.3, 80.1(C3′), 78.4, 77.7, 76.1, 75.8, 75.3, 75.2, 62.8 (C2′), 60.3, 59.1 (C1),32.2 (C2).

To a cooled suspension of Methyltriphenylphosphonium Iodide (0.966 gram,2.7 mmol) in anhydrous THF at 0° C., n-BuLi (1.6 M in hexane, 0.32 mL)was added dropwise and the resulted yellow solution was stirred for anadditional 30 minutes at 0° C. The 6′-aldehyde from the above step(0.822 gram, 1.08 mmol) in anhydrous THF (0.3 mL) was added at 0° C.,and the reaction was allowed to stir for an additional 1.5 hour at roomtemperature. After completion of the reaction as indicated by TLC(EtOAc/Hexane 2:3), the reaction was quenched with saturated NH₄Clsolution. The layers were separated and the aqueous layer was extractedwith ether (2×10 mL). The combined organic layers were washed withbrine, dried over anhydrous MgSO₄ and evaporated to dryness. The crudeproduct was purified by flash chromatography (EtOAc/Hexane 2.5:7.5) toyield Compound 30 (0.4 gram, 50%).

¹H NMR (400 MHz, CDCl₃): ‘Ring I’: δ=H 5.89 (ddd, 1H, J1=17.2, J2=10.4,J3=6.8 Hz, H-6), 5.56 (d, 1H, J=3.9 Hz, H-1), 5.47 (d, 1H, J=17.2 Hz,H-7trans), 5.33-5.27 (m, 1H, H-7cis), 4.64-4.56 (m, 1H, H-5), 4.09 (m,H-3), 3.32-3.27 (m, 2H, H-2, H-4); ‘Ring II’: δ=H 3.69-3.56 (m, 2H, H-4,H-5), 3.54-3.45 (m, 1H, H-1), 3.45-3.35 (m, 2H, H-3, H-6), 2.31 (dt, 1H,J1=13.2, J2=4.5 Hz, H-2eq), 1.49 (ddd, 1H, J1=J2=J3=12.6 Hz, H-2ax); Theadditional peaks in the spectrum were identified as follow: δ=H7.32-7.29 (m, 20H, Bn), 5.02 (d, 1H, J=10.9 Hz, O(CH₂)Bn), 4.94 (dd, 1H,J1=9.9, J2=5.4 Hz, O(CH₂)Bn), 4.89 (d, 1H, J=6.6 Hz, O(CH₂)Bn), 4.83(dd, 2H, J=10.7, 8.5 Hz, O(CH₂)Bn), 4.73 (d, 1H, J=10.9 Hz, O(CH₂)Bn),4.67 (d, 1H, J=10.9 Hz, O(CH₂)Bn), 4.64-4.56 (m, 1H, O(CH₂)Bn).

¹³C NMR (100 MHz, CDCl₃): δ=C 138.2 (Bn), 138.0 (Bn), 138.0 (Bn), 137.4(Bn), 134.9 (Bn), 128.6 (Bn), 128.5 (Bn), 128.5 (Bn), 128.5 (Bn), 128.3(Bn), 128.2 (Bn), 128.1 (Bn), 127.9 (Bn), 127.9 (Bn), 127.7 (Bn), 127.0(Bn), 118.9 (C7′), 97.7 (C1′), 84.7, 84.5, 82.7 (C4′), 79.7 (C3′), 77.7,76.05, 75.6, 75.3, 75.0, 72.7 (C5′), 63.4 (C2′), 60.3 (C1), 59.3, 32.4(C2).

Synthesis of 1-((2R, 3S, 4R, 5R, 6S)-5-azido-3,4-bis(benzyloxy)-6-(((1R,2R, 3S, 4R,6S)-4,6-diazido-2,3-bis(benzyloxy)cyclohexyl)oxy)tetrahydro-2H-pyran-2-yl)-2-(benzyloxy)ethanol(31)

To a stirred solution of Compound 30 (402 mg, 0.53 mmol) in acetone (10mL), water (3 mL) and t-BuOH (10 mL), K2OsO₄.2H₂O (16 mg, 0.051 mmol)and NMO (0.22 mL) were added sequentially. The progress of the reactionwas monitored by TLC (EtOAc/Hexane 2:3), which indicated the completionafter 24 hours. The solvent was thereafter evaporated to dryness; theresidue was dissolved in EtOAc to which an aqueous solution of Na₂S₂O₃was added. The layers were separated and the organic phase was washedwith brine, dried over MgSO₄ and evaporated. The crude product wassubjected to column chromatography (EtOAc/Hexane 1:1) to yielddihydroxylated product (300 mg, 72%) as a 6′-diasteromeric mixture.

A mixture of dihydroxylated compound (0.3 gram, 0.378 mmol) from theabove step and Bu₂SnO (0.103 gram, 0.413 mmol) in toluene/MeOH (10:1, 7mL) was refluxed for 3 hours and concentrated under reduced pressure. Toa solution of this residue in toluene (3 mL) was addedtetrabutylammonium bromide (0.122 gram, 0.378 mmol) and BnBr (0.09 mL,0.756 mmol). The mixture was stirred at 85° C. overnight and quenchedwith addition CH₂Cl₂ (10 mL) and saturated NaHCO₃ (2 mL). Afterfiltration through a pad of Celite®, the organic phase was washed withH₂O (3 mL), brine (5 mL), dried over MgSO₄ and concentrated underreduced pressure. The crude product was purified by columnchromatography (EtOAc/Hexane 2:3) to give the Compound 31 (0.280 gram,84%) as a 6′-diasteromeric mixture.

Synthesis of (1-((2S, 3S, 4R, 5R,6S)-5-azido-3,4-bis(benzyloxy)-6-(((1R, 2R, 3S, 4R,6S)-4,6-diazido-2,3-bis(benzyloxy)cyclohexyl)oxy)tetrahydro-2H-pyran-2-yl)-2-benzyloxy)ethoxy)(tert-butyl)dimethylsilane(32)

Compound 31 (205 mg, 0.232 mmol) was dissolved in anhydrous DMF (5 mL)and cooled to 0° C. t-butyldimethylsilyl chloride (TBSCl, 45 mg, 0.298mmol) was added, followed by addition of Imidazole (39 mg, 0.572 mmol).The reaction mixture was allowed to attain the room temperature understirring, and the reaction progress was monitored by TLC (EtOAc/Hexane3:7). From TLC, reaction did not complete even after prolonged reactiontimes (24 hours) and at this stage the reaction was stopped by addingmixture of ethyl acetate (10 mL) and H₂O (10 mL), and the two layerswere separated. The aqueous layer was thoroughly washed with ethylacetate (4×30 mL). The combined organic layers were washed with sat.NaCl solution and dried over anhydrous MgSO₄. The solvent was evaporatedto dryness and the residue was subjected to column chromatography(EtOAc/Hexane 25:75) to yield corresponding silyl ether (32) (85 mg,23%) as a pure major diastereomer.

Synthesis of1-((2R,3S,4R,5R,6S)-5-azido-3,4-bis(benzyloxy)-6-(((1R,2R,3S,4R, 6S)-4,6-diazido-2,3-bis(benzyloxy)cyclohexyl)oxy)tetrahydro-2H-pyran-2-yl)-2-(benzyloxy)ethanol(31 as pure major diastereomer)

To a stirred solution of Compound 32 (60 mg, 0.06 mmol) in THF (3 mL) atroom temperature, TBAF (0.052 mL, 0.179 mmol) was added and the reactionwas refluxed at 50° C. overnight. After completion of the reaction asindicated by TLC (EtOAc/Hexane 2:3), the solvent was evaporated todryness and the obtained residue was subjected to column chromatography(EtOAc/Hexane 3:7) to yield single diastereromer 31 (52 mg, 95%).

¹H NMR (500 MHz, CDCl₃): ‘Ring I’: δ=H 5.53 (d, 1H, J=3.9 Hz, H-1), 4.17(dd, 1H, J1=10.0, J2=2.4 Hz, H-5), 4.12 (m, 1H, H-6), 3.96 (dd, 1H,J1=10.3, J2=8.9 Hz, H-3), 3.69-3.61 (m, 1H, H-4), 3.50-3.45 (m, 2H, H-7,H-7), 3.22 (dd, 1H, J1=10.3, J2=3.9 Hz, H-2), 3.59 (BrS, 1H, 6′-OH);‘Ring II’: δ=H 3.58-3.49 (m, 2H, H-4, H-5), 3.44-3.11 (m, 3H, H-1, H-3,H-6), 2.23 (dt, 1H, J1=13.2, J2=4.5 Hz, H-2eq), 1.38 (ddd, 1H,J1=J2=J3=12.6 Hz, H-2ax); The additional peaks in the spectrum wereidentified as follow: δ=H 7.29-7.23 (m, 25H, Bn), 4.98 (d, 1H, J=10.8Hz, O(CH₂)Bn), 4.92-4.74 (m, 6H, O(CH₂)Bn), 4.65 (d, 1H, J=11.1 Hz,O(CH₂)Bn), 4.42 (q, 2H, J=11.9 Hz, O(CH₂)Bn).

¹³C NMR (125 MHz, CDCl₃): δ=C 138.0 (Bn), 138.0 (Bn), 137.9 (Bn), 137.7(Bn), 137.3 (Bn), 128.6 (Bn), 128.6 (Bn), 128.5 (Bn), 128.5 (Bn), 128.5(Bn), 128.3 (Bn), 128.1 (Bn), 128.1 (Bn), 128.0 (Bn), 127.9 (Bn), 127.8(Bn), 127.7 (Bn), 127.6 (Bn), 127.0 (Bn), 97.4 (C1′), 84.6, 84.4, 80.8,78.4 (C4′), 77.5, 76.0 (Bn), 75.6 (Bn), 75.3 (Bn), 74.6 (Bn), 73.4 (Bn),71.8, 71.6, 71.2 (C7′), 63.3 (C2′), 60.2 (C1), 59.5 (C3), 32.4 (C2).

Synthesis of (R,X)-Ester

A mixture of (R)-2-methoxy-2(1-naphthyl)propanoic acid [(R)-MαNP] (0.01gram, 0.04 mmol), 4-dimethylaminopyridine (DMAP, 0.006 gram, 0.049mmol), 10-camphorsulfonic acid (CSA, 0.002 gram, 0.008 mmol), and1,3-dicyclohexylcarbodiimide (DCC, 0.047 gram, 0.22 mmol) was stirred inCH₂Cl₂ (3 mL) at 0° C. The major alcohol 31 from the above (0.038 gram,0.043 mmol) was dissolved in CH₂Cl₂ (2 ml), slowly added to the abovestirred mixture, and the reaction was left at room temperature for 72hours. The mixture was diluted with EtOAc and washed with 1% HClsolution, saturated NaHCO₃ and brine. The combined organic layer wasdried over MgSO₄, evaporated and subjected to a column chromatography(EtOAc/Hexane) to yield the desired ester (R,X)-35 (0.008 gram, 17%).

¹H NMR (600 MHz, CDCl₃): ‘Ring I’: δ=H 5.55 (dd, 1H, J=9.9, 3.7 Hz,H-6), 4.87 (d, 1H, J=3.4 Hz, H-1), 3.86 (d, 1H, J=10.0 Hz, H-4),3.50-3.46 (m, 1H, H-7), 3.38 (d, 1H, J=10.2 Hz, H-3), 3.36-3.32 (m, 1H,H-7), 1.55-1.50 (m, 1H, H-5), 1.28 (dd, 1H, J1=10.4, J2=3.9 Hz, H-2),‘Ring II’: δ=H 3.51 (d, 1H, J1=9.7 Hz, H-6), 3.43 (dt, 3H, J1=12.1,J2=7.8 Hz, H-4, H-5, H-3), 3.25 (ddd, 1H, J1=12.6, J2=10.0, J3=4.6 Hz,H-1), 2.23 (dd, 1H, J1=10.9, J2=6.5 Hz, H-2eq), 1.46-1.39 (m, 1H,H-2ax); The additional peaks in the spectrum were identified as follow:6=8.39 (d, 1H, J=8.7 Hz, Ar), 7.80-7.75 (m, 2H, Ar), 7.63 (d, 1H, J=6.4Hz, Ar), 7.54 (t, 2H, J=7.6 Hz, Ar), 7.47-7.40 (m, 2H, Ar), 7.38 (d, 2H,J=7.1 Hz, Ar), 7.37-7.33 (m, 2H, Ar), 7.32-7.27 (m, 9H, Ar), 7.23 (ddd,4H, J1=6.5, J2=4.7, J3=2.2 Hz, Ar), 7.20 (d, 3H, J=8.0 Hz, Ar), 7.09(ddd, 1H, J1=8.5, J2=6.8, J3=1.5 Hz, Ar), 7.06-7.02 (m, 1H, Ar),6.96-6.92 (m, 2H, Ar), 5.01 (d, 1H, J=11.2 Hz, O(CH₂)Bn), 4.88 (d, 2H,J=4.1 Hz, O(CH₂)Bn), 4.84 (d, 1H, J=10.8 Hz, O(CH₂)Bn), 4.59 (d, 1H,J=11.4 Hz, O(CH₂)Bn), 4.50 (d, 1H, J=11.3 Hz, O(CH₂)Bn), 4.44 (d, 1H,J=11.8 Hz, O(CH₂)Bn), 4.25 (d, 1H, J=11.9 Hz, O(CH₂)Bn), 3.99 (d, 1H,J=11.3 Hz, O(CH₂)Bn), 3.71 (d, 1H, J=11.3 Hz, O(CH₂)Bn), 3.07 (s, 1H,OCH₃), 2.02 (s, 3H, CH₃).

¹³C NMR (125 MHz, CDCl₃): δ=C 173.3 (Ar), 138.5 (Ar), 138.4 (Ar), 137.9(Ar), 137.7 (Ar), 137.3 (Ar) 135.3 (Ar), 134.2 (Ar), 131.8 (Ar), 130.1(Ar), 128.9 (Ar), 128.65 (Ar), 128.62 (Ar), 128.5 (Ar), 128.46 (Ar),128.44 (Ar), 128.22 (Ar), 128.22 (Ar), 127.76 (Ar), 127.71 (Ar), 127.5(Ar), 127.4 (Ar), 127.2 (Ar), 126.7 (Ar), 126.4 (Ar), 126.3 (Ar), 126.2(Ar), 124.8 (Ar), 99.7 (C1′), 84.5, 84.43 (s), 81.1, 79.8, 77.0, 76.7,76.1, 75.1, 74.2, 74.2, 73.7, 72.7, 70.2, 69.8, 61.8, 60.2, 59.1, 50.7,32.3, 31.1, 29.8, 21.5 (CH₃).

Synthesis of (S,X)-Ester (36)

A mixture of (S)-2-methoxy-2(1-naphthyl)propanoic acid [(S)-MαNP] (0.007gram, 0.03 mmol), 4-dimethylaminopyridine (DMAP, 0.005 gram, 0.04 mmol),10-camphorsulfonic acid (CSA, 0.001 gram, 0.004 mmol), and1,3-dicyclohexylcarbodiimide (DCC, 0.034 gram, 0.16 mmol) was stirred inCH₂Cl₂ (3 mL) at 0° C. The major alcohol 31 from the above (0.028 gram,0.031 mmol), was dissolved in CH₂Cl₂ (2 ml), slowly added to the abovestirred mixture, and the reaction was left at room temperature for 72hours. The mixture was diluted with EtOAc and washed with 1% HClsolution, saturated NaHCO₃ and brine. The combined organic layer wasdried over MgSO₄, evaporated and subjected to a column chromatography(EtOAc/Hexane) to yield the desired ester (S,X)-36 (0.007 gram, 20%).

¹H NMR (600 MHz, CDCl₃): ‘Ring I’: δ=H 5.49 (dd, 1H, J=8.5, 4.4 Hz,H-6), 5.17 (d, 1H, J=3.8 Hz, H-1), 4.04 (d, 1H, J=10.0 Hz, H-4), 3.58(t, 1H, J=9.8 Hz, H-3), 3.25 (d, 1H, J=8.5 Hz, H-7), 3.22 (dd, 1H,J1=10.7, J2=4.6 Hz, H-7), 2.34 (dd, 1H, J1=17.0, J2=6.3 Hz, H-5),2.12-2.02 (m, 1H, H-2) ‘Ring II’: δ=H 3.51 (dt, 2H, J1=17.8, J2=9.3 Hz,H-4, H-5), 3.46-3.37 (m, 2H, H-1, H-6), 3.33-3.27 (m, 1H, H-3), 2.25(dt, 1H, J1=13.2, J2=4.5 Hz, H-2eq), 1.43 (ddd, 1H, J1=J2=J3=12.6 Hz,H-2ax); The additional peaks in the spectrum were identified as follow:δ=H 8.08 (d, 1H, J=8.8 Hz, Ar), 7.89 (d, 1H, J=7.3 Hz), 7.76 (dd, 2H,J1=15.9, J2=8.1 Hz, Ar), 7.46 (t, 2H, J=7.5 Hz), 7.44-7.41 (m, 1H, Ar),7.39 (d, 1H, J=7.5 Hz, Ar), 7.36 (t, 2H, J=7.3 Hz, Ar), 7.34-7.27 (m,8H, Ar), 7.25-7.23 (m, 2H, Ar), 7.23-7.19 (m, 6H, Ar), 7.16 (t, 1H,J=7.1 Hz, Ar), 7.14-7.09 (m, 3H, Ar), 6.91-6.87 (m, 2H, Ar), 5.01 (d,1H, J=11.1 Hz, O(CH₂)Bn), 4.90-4.79 (m, 3H, O(CH₂)Bn), 4.63 (q, 2H,J=11.1 Hz, O(CH₂)Bn), 4.22-4.15 (m, 2H, O(CH₂)Bn), 4.12 (d, 1H, J=11.0Hz, O(CH₂)Bn), 3.66 (d, 1H, J=11.0 Hz, O(CH₂)Bn), 3.29 (S, 3H, OCH₃),1.97 (s, 3H, CH₃).

¹³C NMR (125 MHz, CDCl₃): δ=C 172.7 (Ar), 138.3 (Ar), 138.0 (Ar), 137.9(Ar), 137.7 (Ar), 137.3 (Ar) 134.1 (Ar), 130.5 (Ar), 129.3 (Ar), 129.1(Ar), 128.6 (Ar), 128.6 (Ar), 128.4 (Ar), 128.4 (Ar), 128.3 (Ar), 128.3(Ar), 128.2 (Ar), 127.9 (Ar), 127.7 (Ar), 127.7 (Ar), 127.6 (Ar), 127.6(Ar), 127.5 (Ar), 126.9 (Ar), 126.1 (Ar), 125.6 (Ar), 125.1 (Ar), 125.1(Ar), 124.6 (Ar), 97.1 (C1′), 84.5 (C5), 84.5 (C4), 81.2, 80.1 (C3′),77.9 (C5′), 77.3, 77.0 (C4), 76.1, 75.2, 74.8, 74.4, 74.3 (C6′), 72.8,70.4 (C4′), 69.4 (C7′), 62.5 (C2′), 60.2 (C3), 59.1 (C1), 51.4 (OCH₃),32.3 (C2), 29.85, 21.9 (CH₃).

The absolute stereochemistry at the 6′ position (denoted by X) was thendetermined by ¹H NMR magnetic anisotropy, which is based on Sector rule7 and relays on the difference in chemical shift values for the assignedprotons in the NMR spectra (see, FIGS. 9A-B). As shown in FIG. 9A, thedifference in chemical shift [Δδ=δ(R, X)-δ(S, X)] for H-5′(−0.82) wasnegative, while that for H-7′, 7′ (+0.23, +0.10) was positive. Accordingto the Sector rule shown in FIG. 9B, the structures (R, X)-MαNP 35 and(S, X)-MaNP 36 are arranged such that OMαNP is positioned on the frontand H-6′ on the back, while the Δδ positive and Δδ negative parts arepositioned on the right and left sides, respectively. These dataconfirms the R configuration (X═R) at the 6′ carbon atom in compound 31.

This study establishes that the major and minor diastereomers, compoundsNB153 and NB155, exhibit (R)- and (S)-configuration at 6′ position:6′-(R)-NB153 and 6′-(S)-NB155.

Example 5 Activity Assays of Exemplary Compounds of Example 4

The experimental assay procedure and result analysis was carried outessentially as described hereinabove and in further detail hereinunder.

Materials and Methods:

In all biological tests, all the tested aminoglycosides were in theirsulfate salt forms [Mw (gr/mol) of the sulfate salts were as follow:Compound 1—437.1, NB74—564.3, NB124—605.9, NB153—526.8, NB155—512.2,NB156—705.9, NB157—746. 6, G418—692.7, gentamicin—653.2].

Dual Luciferase Readthrough Assays:

DNA fragments derived from PCDH15, CFTR, and IDUA cDNAs, including thetested nonsense mutation or the corresponding wild type (wt) codon, andfour to six upstream and downstream flanking codons were created byannealing the following pairs of complementary oligonucleotides:

Usher Syndrome: p.R3Xmut/wt:5′-GATCCCAGAAGATGTTTT/CGACAGTTTTATCTCTGGACAGAGC T-3′ and5′-CTGTCAGAGATAAAACTGTCA/GAAACATCTTCTG-3′; p.R245Xmut/wt:5′GATCCAAAATCTGAATGAGAGGT/CGAACCACCACCACCACCCTCGAG CT-3′ and5′-CGAGGGTGGTGGTGGTTGTTCG/ACCTCTCATTCAGATTTTG-3′; Cystic Fibrosis:p.G542Xmut/wt: 5′-TCGACCAATATAGTTCTTT/GGAGAAGGTGGAATCGAGCT-3′ and and5′-CGATTCCACCTTCTCA/GAAGAACTATATTGG-3′; Hurler Syndrome: p.Q70Xmut/wt:5′-TCGACCCTCAGCTGGGACT/CAGCAGCTCAACCTCGAGCT-3′ and5′-CGAGGTTGAGCTGCTA/GGTCCCAGCTGAGG-3′.

Fragments were inserted in frame into the polylinker of the p2Lucplasmid between either BamHI and SacI (p.R3X and p.R245X) or SalI andSacI (all the rest) restriction sites. For the in vitro readthroughassays, the obtained plasmids, with addition of the testedaminoglycosides were transcribed and translated using the TNTReticulocyte Lysate Quick Coupled Transcription/Translation System.Luciferase activity was determined after 90 minutes of incubation at 30°C., using the Dual Luciferase Reporter Assay System (Promega™). Stopcodon readthrough was calculated as previously described [Grentzmann etal. RNA 1998, 4, 479-486].

Protein Translation Inhibition Tests:

Prokaryotic in vitro translation inhibition by the differentaminoglycosides was quantified in coupled transcription/translationassays by using E. coli S30 extract for circular DNA with the pBESTlucplasmid (Promega), according to the manufacturer's protocol. Translationreactions (25 μL) that contained variable concentrations of the testedaminoglycoside were incubated at 37° C. for 60 minutes, cooled on icefor 5 minutes, and diluted with a dilution reagent (tris-phosphatebuffer (25 mM, pH 7.8), DTT (2 mM), 1,2-diaminocyclohexanetetraacetate(2 mM), glycerol (10%), Triton X-100 (1%) and BSA (1 mg mL-1)) into96-well plates. Eukaryotic in vitro translation inhibition wasquantified by use of TNT® T7 Quick Coupled Transcription/TranslationSystem with a luciferase T7 control DNA plasmid (Promega), according tothe manufacturer protocol. Translation reactions (25 μL) containingvariable concentrations of the tested aminoglycoside were incubated at30° C. for 60 minutes, cooled on ice for 5 minutes, diluted with thedilution reagent and transferred into 96-well plates. In bothprokaryotic and eukaryotic systems the luminescence was measuredimmediately after the addition of the Luciferase Assay Reagent (50 μL;Promega), and the light emission was recorded with a FLx800 FluorescenceMicroplate Reader (Biotek). The half-maximal inhibition concentration(IC50) values were obtained from fitting concentration-response curvesto the data of at least two independent experiments by using Grafit 5software.

Antibacterial Activity Tests:

Comparative antibacterial activities were determined in tworepresentative strains of Gram-negative (E. coli R477-100) andGram-positive (B. subtilis ATCC-6633) bacteria, by measuring the MICvalues using the double-microdilution method according to the NationalCommittee for Clinical Laboratory Standards (NCCLS) (NCCLS. NationalCommittee for Clinical Laboratory Standards, Performance standards forantimicrobial susceptibility testing. Fifth information supplement:Approved Standard M100-S5; Villanova, Pa.: NCCLS, 1994.). All theexperiments were performed in triplicates and analogous results wereobtained in three different experiments.

Results:

FIG. 10 presents comparative plots showing in vitro stop codonsuppression levels induced by Compound 1 (-▪-), NB153 (-▴-), and NB155(-Δ-) in R3X nonsense mutation construct representing USH1 geneticdisease.

These comparative PTC suppression activity tests show that installationof C7′-hydroxyl group (NB153) on Compound 1 dramatically increases itsin vitro readthrough activity, and is more pronounced than the effect ofNB155. These data show an improved activity attributed to the additionalhydroxyl group, and further emphasize the role of stereochemistry at 6′position in RNA target recognition. The observed somewhat higheractivity of NB155 to that of Compound 1 suggests that the additional7′-hydroxyl in NB155 can overcome the configurational penalty at 6′position.

The impact of the additional 7′-hydroxyl in Compounds NB156 and NB157was evaluated against previously published compounds NB74 and NB124,which differ from NB156 and NB157 by the absence of the 7′-hydroxyl, asshown below.

Activity was tested using a collection of dual-luciferase reporterplasmids, containing different sequence contests around premature stopcodons derived from the PCDH15, CFTR, and IDUA genes that underlineUSH1, CF, and MPS I-H, respectively. The exemplified nonsense reporterswere R3X and R245X for USH1, G542X for CF, and Q70X for MPS I-H.

The obtained data is presented in FIGS. 11A-D, showing comparative plotsshowing in vitro stop codon suppression levels induced by NB74 (-Δ-),NB156 (-▴-), and gentamicin (-▪-) (left) and by NB124 (-Δ-), NB157(-▴-), and gentamicin (-▪-) (right), in nonsense constructs representingR3X (USH1) (FIG. 11A), R245X (USH1) (FIG. 11B), Q70X (HS) (FIG. 11C),and G542X (CF) (FIG. 11D). The results are averages of at least threeindependent experiments.

As clearly shown in FIGS. 11A-D, the positive impact of the C7′-hydroxylgroup shown for NB153 is retained also in the pseudo-trisaccharides. Inall mutations tested, the readthrough activity of NB156 is substantiallybetter than that of the structurally related NB74, and the activity ofNB157 is better than its structurally related NB124. In addition, in allmutations tested, the activities of both NB156 and NB157 weresignificantly better than that of the clinical drug gentamicin.

In order to evaluate the specificity toward eukaryotic cytoplasmicribosome, comparative protein translation inhibition of Compounds NB74,NB124, NB156 and NB157 in eukaryotic system was determined, usingcoupled transcription/translation assays.

In all biological tests, all AGs were in their sulfate salt forms, andthe concentrations refer to the free amine form of each AG. Theeukaryotic and prokaryotic half-maximal-inhibition values (IC₅₀ ^(Euk)and IC₅₀ ^(Pro)) were quantified in coupled transcription/translationassays by using active luciferase detection as previously described.Minimal inhibitory concentration (MIC) values were determined by usingthe double-microdilution method.

The obtained data in presented in Table 4 below.

TABLE 4 Antibacterial Activity MIC (μM) Translation Inhibition E. Coli RB. Subtilis Compound IC₅₀ ^(Euk) (μM) IC₅₀ ^(Pro) (μM) 477/100 ATCC6633Gentamicin 62 ± 9  0.03 ± 0.00 6 <0.75 G418 2.0 ± 3   0.01 ± 0.00 9<1.25 Compound 1 347.1 ± 34.3  6.0 ± 1.0 NB153 120.5 ± 14.5  11.0 ±1.2  >311 311 NB155 515.8 ± 15    91.9 ± 8.4  >375 >375 NB74 13.9 ± 1.2 1.0 ± 0.1 680 42 NB156 7.5 ± 0.5 0.7 ± 0.1 >273 34 NB124 1.5 ± 0.1 1.1 ±0.2 1267 156 NB157 1.2 ± 0.1 1.2 ± 0.1 >257 64

The obtained data indicates that the efficacy with which NB157(half-maximal inhibitory concentration value IC₅₀ ^(Euk)=1.2 μM)inhibits eukaryotic translation is greater than that of NB156 (IC₅₀^(Euk)=13.9 μM) and gentamicin (IC₅₀ ^(Euk)=62 μM), similarly to the PTCsuppression activity shown in FIGS. 11A-D. In addition, NB156 and NB157are 1.85-fold and 1.25-fold more specific to the eukaryotic ribosomethan their structurally related Compounds NB74 and NB124, respectively.These data indicate that the elevated PTC suppression activities ofNB156 and NB157 are associated with their increased specificity to theeukaryotic ribosome.

The measured IC₅₀ ^(Pro) and MIC values in Table 4 show that theefficacy with which NB156 and NB157 inhibit the prokaryotic ribosome andtheir subsequent antibacterial activity are very similar to those oftheir structurally related Compounds NB74 and NB124, respectively. Theobserved similar impact on bacterial ribosome by these compounds suggestthat NB156 and NB157 are less ototoxic than gentamicin and G418.

Thus, a new pharmacophoric point, 7′-hydroxyl group, is shown herein asa valuable structural element of the glucosamine ring (Ring I) thatsignificantly affects eukaryotic versus prokaryotic selectivity and thesubsequent PTC suppression activity.

Further assays were conducted essentially as described hereinabove, andsome of the obtained data is presented in FIGS. 12A-13B.

In these assays, the readthrough of a broad arsenal of stop codonmutations in the presence of NB156 and NB157 was tested. Briefly, NB156and NB157 were tested at escalating doses for their read-throughproperties towards the nonsense mutation using the wild-type (WT)sequence of each specific complementary DNA (cDNA) as a control, andplasmids bearing the stop codon mutations in a dual-luciferase assay.DNA fragments derived from different cDNAs were prepared using eitherthe WT or nonsense mutation, in which the sequences from the mutant orwild-type codon were surrounded by four to six upstream and downstreamflanking codons. The cDNA sequence was inserted into the polylinker ofthe p2luc plasmid for each sequence.

The tested mutations and the genetic diseases associated therewith areshown in Table 5 below.

TABLE 5 Mutation Disease G542X Cystic fibrosis R553X Cystic fibrosisW1282X Cystic fibrosis R3381X Duchenne muscular dystrophy Q2522XDuchenne muscular dystrophy mdx Duchenne muscular dystrophy (mouse)W392X Hurler Syndrome Q70X Hurler Syndrome R168X Rett Syndrome R270XRett Syndrome R294X Rett Syndrome R578X Severe epidermolysis bullosaQ251X Severe epidermolysis bullosa R3X Usher Syndrome R245X UsherSyndrome R31X Usher Syndrome

FIG. 12A presents comparative stop-codon mutation readthrough plots,showing percent readthrough as a function of concentration of WT withNB156 (readthrough to 50% renilla), comparing the readthrough of themutations G542X, W392X, R1282X, Q2522X, R3X, Q70X, R578X, R168X, R245X,R31X, mdX, R270X, R3381X, R553X, Q251X and R294X.

FIG. 12B presents comparative stop-codon mutation readthrough plots,showing fold increase of readthrough after exposure to NB156 fromnon-treated control as a function of NB156 concentration, comparing thereadthrough of the mutations G542X, W392X, R1282X, Q2522X, R3X, Q70X,R578X, R168X, R245X, R31X, mdx, R270X, R3381X, R553X, Q251X and R294X.

FIG. 13A presents comparative stop-codon mutation readthrough plots,showing percent readthrough as a function of concentration of WT withNB157 (readthrough to 50% renilla), comparing the readthrough of themutations G542X, W392X, R1282X, Q2522X, R3X, Q70X, R578X, R168X, R245X,R31X, mdX, R270X, R3381X, R553X, Q251X and R294X (see, Table 5 above).

FIG. 13B presents comparative stop-codon mutation readthrough plots,showing fold increase of readthrough after exposure to NB157 fromnon-treated control as a function of NB157 concentration, comparing thereadthrough of the mutations G542X, W392X, R1282X, Q2522X, R3X, Q70X,R578X, R168X, R245X, R31X, mdX, R270X, R3381X, R553X, Q251X and R294X(see, Table 5 above).

In additional comparative assays, the stop-codon mutation readthroughactivity of NB156 was compared to that of NB74. In all tested mutations,NB156 was shown to be more active than NB74.

These data further demonstrate the readthrough activity exhibited byNB156 and NB157 on various stop codon mutations.

Example 6 Unsaturated Glucosamine (Ring I)-Containing ExemplaryCompounds According to Some Embodiments of the Present Invention

Exemplary new modifications of aminoglycoside structures were performedby inserting unsaturation at ring I (glucosamine ring). It has beenassumed that by the deletion of C4′-OH or C3′,C4′-hydroxyls with asimultaneous introduction of unsaturation on Ring I makes the ringrelatively “free” to move within the binding pocket for betterpseudo-pair interaction with G1408 and improved π-π stacking with A1491.

Chemical Syntheses

The following exemplary aminosugars Compounds NB154, NB158 and NB159were synthesized:

All the structures were confirmed and characterized by a combination ofvarious 1D and 2D NMR techniques, including 1D TOCSY, 2D COSY, 2D ¹H-¹³CHMQC and HMBC along with mass spectrometry.

Synthesis of NB154

The synthesis of NB154 is depicted in Scheme 12 below.

Briefly, the synthesis started from paromamine, which is obtained fromcommercially available paromomicin sulfate under acidic (HCl/MeOH)hydrolysis, as previously described. Initially, paromamine was convertedinto the triazide by the action insitu generated triflic azide fromtriflic anhydride and NaN₃ in the presence of CuSO₄ to yield paromamineperazide (18), as described in further detail hereinabove. Uponobtaining the paromamine perazide, the 4′,6′-OH groups were convertedinto corresponding benzylidene acetal (42) using benzaldehyde dimethylacetal under acidic conditions. The other hydroxy groups were convertedto acetate esters in presence of acetic anhydride under basic conditions(43). Deprotection of the arylidene group in Compound 43 under mildacidic environment led to the formation of diol 44 which was subjectedto further post functional transformations to yield the desiredcompound. In order to differentiate the 4′-OH and 6′-OH groups so as toperform selective oxidation, the 6′-OH group in compound 43 wasprotected as its silyl ether 45 by selective protection withtert-butyldiphenylsilyl chloride (TBDPSCl), while the other hydroxylgroup was masked as mesylate ester using mesyl chloride (MsCl) underbase condition (Et₃N) to thereby obtain Compound 46 in excellent yields.

In order to avoid the hydrolysis of 4′-OMs ester functionality duringthe silyl deprotection using TBAF, the TBAF reaction mixture wasbuffered with AcOH and obtained the 6′-OH functional molecule 47 leaving4′-OMs ester intact with the molecule. DMP oxidation of the C-6′hydroxyl group, followed by concomitant elimination of 4′-OMs esterunder basic conditions in one-pot reaction lead to the formation ofcorresponding α, β-unsaturated aldehyde 48 in good yield. Compound 48,upon Luche reduction conditions gave allylic alcohol 49, which ontreatment with NaOMe followed by Staudinger reaction yielded thepseudo-disaccharide NB154.

Synthesis of 1,3,2′-perazido-paromamine (18)

Paromomicin sulfate was hydrolyzed under acidic conditions (HCl/MeOH) toparomamine. Paromamine was converted into the triazide by the in situgenerated triflic azide from triflic anhydride and NaN₃ in the presenceof CuSO₄.

Generation of Triflic azide:

To a vigorously stirred solution of NaN₃ (3.6 grams, 18 equiv.) in water(9.0 mL) and Toluene (9.0 mL) at 0° C., triflic anhydride (4.6 mL, 9.0equiv.) was added drop wise and the reaction mixture was stirred for 30minutes at 0° C. The temperature was thereafter raised to 10° C. and thebiphasic system was stirred for 2 hours. Saturated aqueous NaHCO₃ wasthen added dropwise until the gas evaluation ceased. The phases wereseparated and the aqueous phase was extracted with toluene (2×9 mL). Thecombined organic layers were used in the diazo transfer reaction.

Diazo Transfer Reaction:

Paromamine (1.0 gram, 1.0 equiv.), NaHCO₃ (3.1 grams, 12.0 equiv.) andcopper (II) sulfate were dissolved in water (5.0 mL). Triflic azidestock solution was added, followed by the addition of methanol (40 mL),to thereby obtain a homogeneous solution. The blue color reactionmixture was stirred vigorously at room temperature. Complete conversionof amine was indicated by the change of blue color to green. Afterstirring for 48 hours, TLC (EtOAc/MeOH 95:5) analysis indicated thecompletion of the reaction. The solvent was hereafter evaporated todryness and the residue was subjected to column chromatography (EtOAc100%).

¹H NMR (500 MHz, MeOD): ‘Ring I’: δ_(H)=5.69 (d, 1H, J=3.7 Hz, H-1),3.99 (ddd, 1H, J=9.9, 4.1, 2.6 Hz, H-5), 3.94 (dd, 1H, J=10.2, 9.1 Hz,H-3), 3.84 (dd, 1H, J=11.9, 2.3 Hz, H-6), 3.78 (dd, 1H, J=11.8, 4.4 Hz,H-6), 3.46 (dd, 1H, J=9.7, 9.3 Hz, H-4), 3.13 (dd, 1H, J=10.5, 3.7 Hz,H-2); ‘Ring II’: δ_(H)=3.80 (t, 1H, J=8.8 Hz, H-5), 3.77-3.67 (m, 3H,H-1, H-3, H-4), 3.56 (t, 1H, J=9.6 Hz, H-6), 2.59-2.48 (m, 1H), 1.68(dd, 1H, J=26.3, 12.7 Hz, H-2).

¹³C NMR (125 MHz, MeOD): δ_(C)=99.3 (C1′), 80.7, 77.8 (C5), 77.7 (C6),73.9 (C5′), 72.4 (C3′), 71.6, 64.8 (C2′), 62.1 (C6′), 61.6, 60.9, 33.1(C2).

MALDI TOFMS: calculated for C₁₂H₁₉N₉O₇ ([M+K]⁺) m/e 440.3; measured m/e440.2).

Preparation of 4′,6′-O-benzylidene-1,2′,3-triazido-paromamine (42

Compound 18 (1 gram, 2.49 mmol) was dissolved in dry DMF (20 mL) andBenzaldehyde dimethyl acetal (0.87 mL, 5.79 mmol) and a catalytic amountof CSA were added. The reaction mixture was stirred at 60° C. and thereaction progress was monitored by TLC (EtOAc 60%, Hexane 40%), whichindicated the completion of the reaction after 2 hours. The reactionmixture was diluted with EtOAc and extracted with saturated aqueoussolutions of NaHCO₃ and Brine. The combined organic layer was dried overMgSO₄, filtered and concentrated under reduced pressure. The crudeproduct was purified by flash chromatography (EtOAc/hexane 1:1) toafford Compound 42 (1.0 gram, 8 3% yield).

¹H NMR (600 MHz, MeOD): ‘Ring I’: δ_(H)=5.69 (d, 1H, J=3.5 Hz, H-1),4.27 (dd, 1H, J₁=10.0, J₂=5.0 Hz, H-6), 4.20 (td, 1H, J₁=10.1, J₂=5.0Hz, H-6), 4.15 (t, 1H, J=9.7 Hz, H-3), 3.81 (t, 1H, J=10.1 Hz, H-5),3.59 (t, 1H, J=9.56 Hz, H-4), 3.31 (dd, 1H, J₁=10.4, J₂=4.6 Hz, H-2);‘Ring II’: δ_(H)=3.57 (t, 1H, J=8.2 Hz, H-5), 3.54-3.48 (m, 2H, H-3,H-4), 3.45 (ddd, 1H, J=14.7, 11.3, 5.5 Hz, H-1), 3.31 (t, 1H, J=9.7 Hz,H-6), 2.28 (dt, 1H, J₁=8.5, J₂=3.9 Hz, H-2eq), 1.46 (ddd, 1H,J₁=J₂=J₃=12.3 Hz, H-2ax); the additional peaks in the spectrum wereidentified as follow: 7.58-7.50 (m, 2H), 7.43-7.35 (m, 3H, Ar),7.43-7.35 (m, 3H, Ar), 5.63 (s, 1H, PhCH).

¹³C NMR (150 MHz, MeOD): &c=139.10 (Ar), 129.98 (Ar), 129.07 (Ar),127.56 (Ar), 103.14 (PhCH), 100.36 (C-1′), 83.06, 81.33, 77.82, 77.81,69.85 (C-5′), 69.59 (C-6′), 65.23 (s), 64.58 (s), 61.76 (s), 60.95 (s),33.21 (C-2).

MALDI TOFMS: calculated for C₁₉H₂₄N₉O₇ ([M+H]+) m/e 490.4; measured m/e490.0.

Preparation of 4′,6′-O-benzylidene-1,2′,3-triazido-peracetylparomamine(43)

Compound 42 (1.4 gram, 2.94 mmol) was dissolved in anhydrous pyridine (8mL) and Acetic anhydride (1.4 mL, 14.8 mmol), and 4-DMAP (3.2 grams,26.1 mmol) was added. The reaction progress was monitored by TLC, whichindicated completion after 4 hours. The reaction mixture was dilutedwith EtOAc, and extracted with aqueous solution of HCl (2%), saturatedaqueous NaHCO₃, and brine. The combined organic layers were dried overanhydrous MgSO₄ and concentrated. The crude product was purified bysilica gel column chromatography (EtOAc/Hexane 4:6) to afford 43 (1.32gram, 73% yield).

¹H NMR (600 MHz, MeOD): ‘Ring I’: δ_(H)=5.57 (dd, 1H, J=10.3, J₂=9.6 Hz,H-3), 5.15 (d, 1H, J=3.2 Hz, H-1), 4.31 (dt, 2H, J₁=13.0, J₂=5.0 Hz,H-5, H-6), 3.73 (dd, 1H, J₁=14.4, J₂=5.6 Hz, H-6), 3.62 (t, 1H, J=9.3Hz, H-4), 3.24 (dd, 1H, J=10.5, J₂=4.0 Hz, H-2); ‘Ring II’: δ_(H)=5.17(t, 1H, J=9.7 Hz, H-5), 4.92 (t, 1H, J=10.0 Hz, H-6), 3.74-3.56 (m, 2H,H-4, H-1), 3.46 (ddd, 1H, J=12.2, J2=10.1, J₃=4.9 Hz, H-3), 2.43 (dt, 1HJ₁=13.0, J₂=4.5 Hz, H-2), 1.59 (ddd, 1H, J=25.8, J₂=12.8 Hz, H-2); theadditional peaks in the spectrum were identified as follow: δ_(H)=7.44(dt, J₁=5.0, J₂=3.0 Hz, 2H, Ar), 7.39-7.30 (m, 3H, Ar), 5.49 (s, 1H,PhCH).

¹³C NMR (150 MHz, CDCl₃): δ_(c)=170.06 (C═O), 169.76 (C═O), 169.37(C═O), 136.93 (Ar), 129.26 (Ar), 128.36 (Ar), 126.30 (Ar), 101.74(PhCH), 100.22 (C-1′), 79.17 (C-4′), 78.72 (C-4), 74.27 (C-6), 73.72(C-5), 68.69 (C-6′), 68.63 (C-3′), 63.51 (C-5′), 61.46 (C-2′), 58.29(C-3), 57.68 (C-1), 31.77 (C-2), 20.87 (CH₃CO), 20.67 (CH₃CO), 20.64(CH₃CO).

Preparation of(1S,2S,3R,4S,6R)-3-(((2S,3R,4R,5S,6R)-4-acetoxy-3-azido-5-hydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-4,6-diazidocyclohexane-1,2-diyl diacetate (44)

Compound 43 (1.32 gram, 2.14 mmol) was dissolved in mixture of AcOH/H₂O(5:1, 10 mL) and the solution was stirred at 60° C. overnight. After thereaction completion, as indicated by TLC, the aqueous acetic acid wasremoved by evaporation. The crude residue was dissolved in EtOAc, andextracted with saturated aqueous NaHCO₃, and brine. The combined organiclayers were dried over anhydrous MgSO₄ and concentrated. The crudeproduct was purified by silica gel column chromatography (EtOAc/Hexane6:4) to afford 44 (771 mg, 68% yield).

¹H NMR (600 MHz, CDCl₃): ‘Ring I’: δ_(H)=5.28 (t, 1H, J=9.9 Hz, H-3),5.13 (d, 1H, J=3.6 Hz, H-1), 4.09 (d, 1H, J=10.0 Hz, H-4), 3.95-3.79 (m,2H, H-6, H-6), 3.67 (t, 1H, J=9.1 Hz, H-5), 3.28 (dd, 1H, J₁=10.3,J₂=3.5 Hz, H-2); ‘Ring II’: δ_(H)=5.12 (t, 1H, J=9.8 Hz, H-5), 4.91 (t,1H, J=10.0 Hz, H-6), 3.73-3.67 (m, 1H, H-3), 3.63 (t, 1H, J=9.7 Hz,H-4), 3.52 (td, 1H, J=12.1, 4.6 Hz, H-1), 2.42 (dt, 1H, J 1=13.2, J2=4.4 Hz, H-2), 1.59 (ddd, 1H, J₁=J₂=J₃=12.6 Hz, H-2); the additionalpeaks in the spectrum were identified as follow: 6=2.15 (s, 3H, CH₃C═O),2.11-2.02 (m, 6H, CH₃C═O).

¹³C NMR (150 MHz, CDCl₃): δ_(c)=171.78 (C═O), 170.10 (C═O), 169.73(C═O), 99.33 (C-1′), 78.79 (C-4), 74.21 (C-6), 73.68 (C-5), 73.03(C-3′), 72.62 (C-4′), 69.45 (C-5′), 61.64 (C-6′), 61.02 (C-2′), 58.71(C-1), 57.65 (C-3), 31.98 (C-2), 21.06 (CH₃CO), 20.72 (CH₃CO), 20.66(CH₃CO).

Preparation of(1S,2S,3R,4S,6R)-3-(((2S,3R,4S)-4-acetoxy-3-azido-6-formyl-3,4-dihydro-2H-pyran-2-yl)oxy)-4,6-diazidocyclohexane-1,2-diyl Diacetate (48)

To a stirred solution of compound 47 (88 mg, 0.145 mmol) in CH₂Cl₂ (3mL) at 0° C., DMP (123 mg, 0.289 mmol) was added in one portion and theresulting mixture was stirred at 0° C. for 40 minutes. Then the reactionmixture was allowed to reach room temperature and stirred for additional3 hours. After completion of the reaction as indicated by TLC, Et₃N (0.2mL) was added in one-pot at r.t. and mixture was stirred 30 minutes.Thereafter, the reaction mixture was diluted with EtOAc and washed withwater, followed by brine. The combined organic layers were dried overanhydrous MgSO₄ and concentrated. The crude product was purified bysilica gel column chromatography (EtOAc/Hexane 3:7) to afford 48 (50 mg,68% yield).

¹H NMR (600 MHz, CDCl₃): ‘Ring I’: δ_(H)=5.93 (d, 1H, J=2.6 Hz, H-4),5.76 (dd, 1H, J₁=9.4, J₂=2.4 Hz, H-3), 5.38 (d, 1H, J=2.6 Hz, H-1), 3.71(dd, 1H, J₁=9.4, J₂=2.7 Hz, H-2); ‘Ring II’: δ_(H)=5.1³ (t, 1H, J=9.9Hz, H-5), 4.90 (t, 1H, J=10.0 Hz, H-6), 3.82 (t, 1H, J=9.8 Hz, H-4),3.60 (ddd, 1H, J₁=12.6, J₂=10.2, J₃=4.6 Hz, H-1), 3.42 (ddd, 1H,J₁=12.6, J₂=10.0, J₃=4.6 Hz, H-3), 2.31 (dt, 1H, J₁=13.5, J₂=4.6 Hz,H-2), 1.49 (ddd, 1H, J₁=J₂=J₃=12.7 Hz, H-2); the additional peaks in thespectrum were identified as follow: 6=9.24 (s, 1H, CHO), 2.14 (s, 3H,CH₃CO), 2.08 (s, 3H, CH₃CO), 2.06 (s, 3H, CH₃CO);

¹³C NMR (150 MHz, CDCl₃): δ_(c)=185.12 (CHO), 170.01 (C═O), 169.87(C═O), 169.48 (C═O), 148.79 (C-5′), 116.71 (C-4′), 98.98 (C-1′), 79.20(C-4), 73.99 (C-6), 73.25 (C-5), 66.43 (C-3′), 59.14 (C-3), 58.50(C-2′), 57.84 (C-1), 32.14 (C-2), 20.91 (CH₃CO), 20.69 (CH₃CO), 20.64(CH₃CO).

Preparation of(1S,2S,3R,4S,6R)-3-(((2S,3R,4S)-4-acetoxy-3-azido-6-(hydroxymethyl)-3,4-dihydro-2H-pyran-2-yl)oxy)-4,6-diazidocyclohexane-1,2-diyl diacetate (49)

To a stirred solution of aldehyde 48 (1.0 gram, 1.97 mmol) in dry MeOH(10 mL), cooled to 0° C., CeCl₃.7H₂O (734 mg, 1.97 mmol) and NaBH₄ (74mg, 1.95 mmol) were added successively. The progress of the reaction wasmonitored by TLC (EtOAc/Hexane 2:3), which indicated completion after 1hour. The MeOH was evaporated completely and H₂O was added. The aqueouslayer was extracted with EtOAc. The combined organic layers were washedwith brine, dried over MgSO₄, evaporated to dryness, and purified bycolumn chromatography (silica gel, EtOAc/Hexane) to yield correspondingallyl alcohol 49 (960 mg, 96%).

¹H NMR (600 MHz, CDCl₃): ‘Ring I’: δ_(H)=5.44 (d, 1H, J=5.9, H-3), 5.25(d, 1H, J=2.4 Hz, H-1), 5.03 (d, 1H, J=2.7 Hz, H-4), 4.09-3.96 (m, 2H,H-6, H-6), 3.58 (dd, 1H, J₁=7.0, J₂=2.5 Hz, H-2); ‘Ring II’: δ_(H)=5.12(t, 1H, J=9.9 Hz, H-5), 4.89 (t, 1H, J=10.0 Hz, H-6), 3.79 (t, 1H, J=9.8Hz, H-4), 3.69-3.54 (m, 1H, H-1), 3.48 (ddd, 1H, J₁=12.6, J2=10.0,J₃=4.6 Hz, H-3), 2.30 (dt, 1H, J₁=13.4, J₂=4.5 Hz, H-2eq), 1.44 (ddd,1H, J₁=J2=J3=12.8 Hz, H-2ax); The additional peaks in the spectrum wereidentified as follow: δ_(H)=2.57 (brs, 1H, 6′-OH), 2.06 (s, 3H, CH₃),2.04 (s, 6H, CH₃).

¹³C NMR (150 MHz, CDCl₃): δ_(C)=170.0 (CH₃—CO), 169.9 (CH₃—CO), 169.4(CH₃—CO), 152.6 (C5′), 98.3 (C1′), 96.3 (C4′), 78.8 (C4), 73.9 (C6),73.3 (C5), 66.6 (C3′), 61.7 (C6′), 59.3 (C3), 58.9 (C2′), 57.8 (C1),32.3 (C2), 21.0 (CH₃), 20.6 (CH₃), 20.5 (CH₃).

Preparation of (1S,2R,3R,4S, 6R)-4,6-diazido-3-(((2S,3R,4S)-3-azido-4-hydroxy-6-(hydroxymethyl)-3,4-dihydro-2H-pyran-2-yl)oxy)cyclohexane-1,2-diol(50)

To a stirred solution of alcohol 49 under argon atmosphere (960 mg, 1.88mmol) in dry MeOH (15 mL), NaOMe (459 mg, 8.49 mmol) was added. Theprogress of the reaction was monitored by TLC (EtOAc/Hexane 3:2), whichindicated completion after 6 hours. Then the reaction mixture was passedthrough a pad of silica gel column and the column was washed with MeOH.The combined organic layers were evaporated to dryness, and purified bycolumn chromatography (silica gel, EtOAc/Hexane) to yield compound 50(700 mg, 97%).

¹H NMR (500 MHz, MeOD): ‘Ring I’: δ_(H)=5.80 (d, 1H, J=2.5 Hz, H-1),5.03 (dt, 1H, J₁=2.5, J₂=1.0 Hz, H-4), 4.47-4.39 (m, 1H, H-3), 4.06-3.96(m, 2H, H-6), 3.42 (dd, 1H, J₁=8.0, J₂=2.5 Hz, H-2); ‘Ring II’:δ_(H)=3.61 (t, 1H, J=9.5 Hz, H-4), 3.52 (t, 1H, J=9.5 Hz, H-5), 3.46(ddd, 1H, J₁=12.5, J2=9.5, J₃=4.5 Hz, H-3), 3.43-3.37 (m, 1H, H-1), 3.26(t, 1H, J=9.5 Hz, H-6), 2.16 (dt, 1H, J₁=12.5, J₂=4.5 Hz, H-2eq), 1.29(ddd, 1H, J₁=J₂=J₃=12.5 Hz, H-2ax).

¹³C NMR (125 MHz, MeOD): δ_(C)=152.6, 100.5 (C4′), 99.6 (C1′), 81.7(C4), 77.9 (C6), 77.6 (C5), 64.9 (C3′), 63.8 (C2′), 62.0 (C1), 61.9(C6′), 61.6 (C3), 33.7 (C2).

MALDI TOFMS: calculated for C₁₂H₁₇N₉O₆ ([M+Na]⁺) m/e 406.3; measured m/e406.3.

Preparation of(1S,2R,3R,4S,6R)-4,6-diamino-3-(((2S,3R,4S)-3-amino-4-hydroxy-6-(hydroxymethyl)-3,4-dihydro-2H-pyran-2-yl)oxy)cyclohexane-1,2-diol(NB154)

To a stirred solution of Compound 50 (256 mg, 1.0 equiv.) in a mixtureof THF (3.0 mL) and aqueous NaOH (1 mM, 5.0 mL), PMe₃ (1 M solution inTHF, 0.55 mL, 7.8 equiv.) was added. The progress of the reaction wasmonitored by TLC [CH₂Cl₂/MeOH/H₂O/MeNH₂ (33% solution in EtOH),10:15:6:15], which indicated completion after 3.5 hours. The reactionmixture was purified by flash chromatography on a short column of silicagel. The column was washed with the following solvents: THF (100 mL),CH₂Cl₂ (100 mL), EtOH (50 mL), and MeOH (100 mL). The product was theneluted with the mixture of 5% MeNH₂ solution (33% solution in EtOH) in80% MeOH. Fractions containing the product were combined and evaporatedunder vacuum. The pure product was obtained by passing the above productthrough a short column of Amberlite CG50 (NH₄ form). First, the columnwas washed with water, then the product was eluted with a mixture of 10%NH₄OH in water to yield NB154 (184 mg, 90%).

For storage and biological tests, NB154 was converted to its sulfatesalt form as follow: The free base form was dissolved in water, the pHwas adjusted to 7 with H₂SO₄ (0.1 N) and lyophilized to afford thesulfate salt of NB154.

¹H NMR (500 MHz, MeOD): ‘Ring I’: δ_(H)=5.40 (d, 1H, J=2.5 Hz, H-1),4.98 (d, 1H, J=3.0 Hz, H-4), 4.06 (dd, 1H, J₁=7.0, J₂=3.0 Hz, H-3),4.01-3.91 (m, 2H, H-6), 2.92 (dd, 1H, J₁=7.0, J₂=2.5 Hz, H-2); ‘RingII’: δ_(H)=3.41-3.35 (m, 2H, H-4, H-5), 3.09 (t, 1H, J=9.5 Hz, H-6),2.76-2.70 (m, 1H, H-3), 2.66 (ddd, 1H, J₁=12.5, J₂=10.0, J₃=4.5 Hz,H-1), 2.03 (dt, 1H, J₁=12.5, J₂=4.5 Hz, H-2eq), 1.24 (ddd, 1H,J₁=J₂=J₃=12.5 Hz, H-2ax).

¹³C NMR (125 MHz, MeOD): δ_(C)=152.6, 101.8 (C1′), 101.5 (C4′), 86.7,78.8 (C6), 77.7, 68.0 (C3′), 62.5 (C6′), 55.6 (C2′), 52.4 (C3), 51.2(C1), 36.6 (C2).

MALDI TOFMS: calculated for C₁₂H₂₃N₃O₆ ([M+H]⁺) m/e 306.3; measured m/e306.8.

Syntheses of NB158 and NB159

NB158 and NB159 were prepared as depicted in Scheme 13.

Briefly, the syntheses of pseudo-trisaccharides NB158 and NB159 wereaccomplished from the corresponding acceptor 51, which is obtained fromregioselective acetylation of 50 at low temperature (−20° C.) usingacetic anhydride in pyridine. Acceptor 51 upon glycosylation reactionwith trichloroacetimidate donors 52 and 53 with catalytic amount ofBF₃—OEt₂ afforded the protected pseudo-trisaccharides 54 and 55exclusively as corresponding p3-anomers in excellent yields. The globalester deprotection of pseudo-trisaccharides 54 and 55 with methylamineand the Staudinger reaction to convert azides into corresponding aminesresulted in Compounds NB158 and NB159.

Preparation of((2S,3R,4S)-4-acetoxy-2-(((1R,2S,3S,4R,6S)-3-acetoxy-4,6-diazido-2-hydroxycyclohexyl)oxy)-3-azido-3,4-dihydro-2H-pyran-6-yl)methylacetate (51)

Compound 50 (700 mg, 1.82 mmol) was dissolved in anhydrous pyridine (8mL) and cooled to −20° C. At this temperature, acetic anhydride (0.6 mL,6.19 mmol) was added dropwise and the reaction was allowed to progressat −20° C. The reaction progress was monitored by TLC, which indicatedcompletion after 17 hours. The reaction mixture was diluted with EtOAc,and extracted with aqueous solution of NaHCO₃, HCl (2%), saturatedaqueous NaHCO₃, and brine. The combined organic layers were dried overanhydrous MgSO₄ and concentrated. The crude product was purified bysilica gel column chromatography to afford 51 (520 mg, 56%).

¹H NMR (600 MHz, CDCl₃): ‘Ring I’: δ_(H)=5.62 (d, 1H, J=8.7, H-3), 5.59(d, 1H, J=2.8 Hz, H-1), 5.03 (d, 1H, J=2.7 Hz, H-4), 4.52 (q, 2H, J=13.4Hz, H-6, H-6), 3.77 (dd, 1H, J₁=8.7, J₂=2.8 Hz, H-2); ‘Ring II’:δ_(H)=4.86 (t, 1H, J=9.9 Hz, H-6), 3.69 (td, 1H, J=9.5, J₂=4.3 Hz, H-5),3.58 (t, 1H, J=9.5 Hz, H-4), 3.50 (ddd, 1H, J₁=12.6, J₂=10.0, J₃=4.6 Hz,H-1), 3.37 (ddd, 1H, J₁=12.6, J₂=9.8, J₃=4.6 Hz, H-3), 2.28 (dt, 1H,J₁=13.5, J2=4.6 Hz, H-2eq), 1.43 (ddd, 1H, J₁=J₂=J₃=12.6 Hz, H-2ax); Theadditional peaks in the spectrum were identified as follow: δ_(H)=2.17(s, 3H, CH₃), 2.12 (s, 3H, CH₃), 2.10 (s, 3H, CH₃).

¹³C NMR (150 MHz, CDCl₃): δ_(C)=170.9 (CH₃—CO), 170.4 (CH₃—CO), 170.4(CH₃—CO), 148.2 (C5′), 99.1 (C4′), 98.8 (C1′), 83.1 (C4), 75.7 (C6),74.7 (C5), 67.4 (C3′), 62.4 (C6′), 59.7 (C2′), 59.1 (C3), 58.0 (C1),32.6 (C2), 21.1 (CH₃), 20.9 (CH₃), 20.9 (CH₃).

Preparation of Glycosylation Product (54)

Anhydrous CH₂Cl₂ (15 mL) was added to a powdered, flame-dried 4 Åmolecular sieves (2.0 grams), followed by the addition of acceptor 51(270 mg, 0.53 mmol) and donor 52 (1.115 gram, 2.11 mmol). The reactionmixture was stirred for 10 minutes at room temperature and was thencooled to −30° C. At this temperature, catalytic amount of BF₃.Et₂O (0.1ml) was added and the mixture was stirred at −30° C. and the reactionprogress was monitored by TLC, which indicated the completion after 60minutes. The reaction mixture was diluted with ethyl acetate and washedwith saturated NaHCO₃ and brine. The combined organic layer was driedover MgSO₄, evaporated and subjected to column chromatography(EtOAc/Hexane) to obtain Compound 54 (370 mg) in 80% yield.

¹H NMR (600 MHz, CDCl₃): ‘Ring I’: δ_(H)=5.69 (d, 1H, J=2.3, H-1), 5.43(dd, 1H, J₁=6.4, J₂=4.0 Hz, H-3), 5.07 (d, 1H, J=3.3 Hz, H-4), 4.55 (q,2H, J=13.3 Hz, H-6, H-6), 3.92 (dd, 1H, J₁=6.8, J₂=2.3 Hz, H-2); ‘RingII’: δ_(H)=5.0 (t, 1H, J=10.1 Hz, H-6), 3.87 (t, 1H, J=9.4 Hz, H-5),3.79 (t, 1H, J=9.6 Hz, H-4), 3.49 (ddd, 1H, J₁=12.2, J₂=10.0, J₃=4.3 Hz,H-1), 3.43 (ddd, 1H, J₁=12.1, J₂=9.8, J₃=4.5 Hz, H-3), 2.34-2.22 (m, 1H,H-2eq), 1.45 (ddd, 1H, J₁=J2=J3=12.7 Hz, H-2ax); ‘Ring III’: δ_(H)=5.56(d, 1H, J=1.1 Hz, H-1), 5.55-5.53 (m, 1H, H-2), 5.44 (dd, 1H, J₁=6.8,J2=5.3 Hz, H-3), 4.57-4.49 (m, 1H, H-4), 3.66 (dd, 1H, J₁=13.5, J₂=3.6Hz, H-5), 3.56 (dd, 1H, J₁=13.3, J₂=6.0 Hz, H-5); The additional peaksin the spectrum were identified as follow: δ_(H)=7.93 (t, 2H, J=4.2 Hz,Ar), 7.88 (dd, 2H, J₁=8.3, J2=1.2 Hz, Ar), 7.59-7.50 (m, 2H, Ar), 7.39(t, 2H, J=7.9 Hz, Ar), 7.34 (t, 2H, J=7.9 Hz, Ar), 2.29 (s, 3H, CH₃),2.10 (s, 3H, CH₃), 2.09 (s, 3H, CH₃).

¹³C NMR (150 MHz, CDCl₃): δ_(C)=170.3 (CH₃—CO), 170.1 (CH₃—CO), 170.0(CH₃—CO), 165.5 (C₆H5-CO), 165.2 (C₆H5-CO), 149.3 (C5′), 133.8 (Ar),133.7 (Ar), 129.7 (Ar), 129.7 (Ar), 128.8 (Ar), 128.7 (Ar), 128.6 (Ar),128.5 (Ar), 107.5 (C1″), 97.9 (C1′), 97.8 (C4′), 80.8 (C4″, C4), 78.9(C5), 74.7 (C2″), 73.9 (C6), 71.7 (C3′), 66.8 (C3″), 62.3 (C6′), 59.8(C3), 59.3 (C2′), 58.4 (C1), 52.7 (C5″), 32.5 (C2), 21.1 (CH₃), 20.9(CH₃), 20.8 (CH₃).

Preparation of Compound 56

The glycosylation product 54 (370 mg, 0.422 mmol) was treated with asolution of MeNH₂ (33% solution in EtOH, 15 mL) and the reactionprogress was monitored by TLC (EtOAc/MeOH 85:15), which indicatedcompletion after 12 hours. The reaction mixture was evaporated todryness and was subjected to column chromatography (MeOH/EtOAc 2:8) toobtain the corresponding completely unprotected perazido derivative 56(237 mg) in 97% yield.

¹H NMR (600 MHz, MeOD): ‘Ring I’: δ_(H)=5.83 (d, 1H, J=2.5, H-1), 5.02(dd, 1H, J₁=1.8, J₂=1.1 Hz, H-4), 4.35 (dd, 1H, J₁=4.4, J₂=2.4 Hz, H-3),4.05-3.94 (m, 2H, H-6, H-6), 3.53 (dd, 1H, J₁=7.6, J₂=4.2 Hz, H-2);‘Ring II’: δ_(H)=3.70 (t, 1H, J=9.7 Hz, H-4), 3.62 (t, 1H, J=9.1 Hz,H-5), 3.49-3.41 (m, 1H, H-3), 3.39 (dt, 1H, J₁=9.8, J₂=4.9 Hz, H-1),3.37-3.34 (m, 1H, H-6), 2.12 (dt, 1H, J₁=13.0, J₂=4.5 Hz, H-2eq), 1.23(ddd, 1H, J₁=J2=J₃=12.5 Hz, H-2ax); ‘Ring III’: δ_(H)=5.37 (d, 1H, J=1.3Hz, H-1), 4.16 (dd, 1H, J₁=4.7, J₂=1.3 Hz, H-2), 4.10 (dd, 1H, J₁=7.7,J₂=4.2 Hz, H-3), 4.02 (dd, 1H, J₁=7.0, J₂=2.7 Hz, H-4), 3.59 (dd, 1H,J₁=13.3, J₂=3.2 Hz, H-5), 3.50 (dd, 1H, J₁=13.2, J₂=6.4 Hz, H-5);

¹³C NMR (150 MHz, MeOD): δ_(C)=152.8 (C5′), 111.1 (C1″), 100.1 (C4′),98.8 (C1′), 83.9 (C5), 82.4 (C4″), 79.7 (C4), 77.5 (C6), 76.2 (C2″),72.4 (C3″), 65.3 (C3′), 64.0 (C2′), 62.1 (C6′), 61.9 (C1), 61.7 (C3),54.2 (C5″), 33.5 (C2).

Preparation of NB158

To a stirred solution of compound 56 (237 mg, 0.438 mmol) in a mixtureof THF (3 mL) and aqueous NaOH (1 mM, 5 mL), PMe₃ (1 M solution in THF,3.5 mL, 40.1 mmol) was added. The progress of the reaction was monitoredby TLC [CH₂Cl₂/MeOH/H₂O/MeNH₂ (33% solution in EtOH), 10:15:6:15], whichindicated completion after 3 hours. The reaction mixture was purified byflash chromatography on a short column of silica gel. The column waswashed with the following solvents: THF (100 mL), CH₂Cl₂ (100 mL), EtOH(50 mL), and MeOH (100 mL). The product was then eluted with the mixtureof 5% MeNH₂ solution (33% solution in EtOH) in 80% MeOH. Fractionscontaining the product were combined and evaporated under vacuum. Thepure product was obtained by passing the above product through a shortcolumn of Amberlite CG50 (NH₄ form). First, the column was washed withwater, then the product was eluted with a mixture of 10% NH₄OH in waterto yield NB158 (138 mg, 75%).

For storage and biological tests, NB158 was converted to its sulfatesalt form as follow: The free base form was dissolved in water, the pHwas adjusted to 6.7 with H₂SO₄ (0.1 N) and lyophilized to afford thesulfate salt of NB158.

¹H NMR (600 MHz, MeOD): ‘Ring I’: δ_(H)=5.40 (d, 1H, J=2.0, H-1), 5.01(d, 1H, J=3.7 Hz, H-4), 4.04 (t, 1H, J=5.3 Hz, H-3), 4.0 (s, 2H, H-6,H-6), 3.09 (dd, 1H, J₁=5.1, J₂=1.9 Hz, H-2); ‘Ring II’: δ_(H)=3.57-3.50(m, 2H, H-4, H-5), 3.19 (t, 1H, J=9.1 Hz, H-6), 2.79 (ddd, 1H, J₁=12.5,J2=9.3, J3=4.3 Hz, H-3), 2.67 (ddd, 1H, J₁=11.8, J₂=9.9, J₃=4.1 Hz,H-1), 2.04 (dt, 1H, J₁=8.3, J₂=6.2 Hz, H-2eq), 1.24 (ddd, 1H,J₁=J2=J3=12.3 Hz, H-2ax); ‘Ring III’: δ_(H)=5.29 (s, 1H, H-1), 4.14 (d,1H, J=5.4 Hz, H-2), 4.06-4.02 (m, 1H, H-3), 3.92-3.87 (m, 1H, H-4), 2.98(dd, 1H, J₁=13.0, J₂=4.4 Hz, H-5), 2.84 (dd, 1H, J₁=12.9, J₂=8.4 Hz,H-5);

¹³C NMR (150 MHz, MeOD): δ_(C)=153.4 (C5′), 110.6 (C1″), 100.5 (C4′,C1′), 84.9 (C5), 84.45 (C4), 84.41 (C4″), 78.9 (C6), 76.3 (C2″), 72.8(C3″), 67.8 (C3′), 62.3 (C6′), 55.0 (C2′), 52.5 (C1), 51.4 (C3), 45.4(C5″), 37.2 (C2).

Preparation of Glycosylation product (55)

Anhydrous CH₂Cl₂ (15 mL) was added to a powdered, flame-dried 4 Åmolecular sieves (2.0 grams), followed by the addition of acceptor 51(265 mg, 0.520 mmol) and donor 53 (1.12 gram, 2.06 mmol). The reactionmixture was stirred for 10 minutes at room temperature and was thencooled to −30° C. At this temperature, catalytic amount of BF₃.Et₂O (0.1ml) was added and the mixture was stirred at −30° C. and the reactionprogress was monitored by TLC, which indicated the completion after 60minutes. The reaction mixture was diluted with ethyl acetate and washedwith saturated NaHCO₃ and brine. The combined organic layer was driedover MgSO₄, evaporated and subjected to column chromatography(EtOAc/Hexane) to obtain Compound 55 (295 mg) in 64% yield.

¹H NMR (600 MHz, CDCl₃): ‘Ring I’: δ_(H)=5.69 (d, 1H, J=2.4, H-1), 5.42(dd, 1H, J₁=6.7, J₂=3.8 Hz, H-3), 5.06 (d, 1H, J=3.0 Hz, H-4), 4.54 (q,2H, J=13.3 Hz, H-6, H-6), 3.96 (dd, 1H, J₁=6.8, J₂=2.5 Hz, H-2); ‘RingII’: δ_(H)=4.99 (t, 1H, J=9.9 Hz, H-6), 3.87 (t, 1H, J=9.5 Hz, H-5),3.78 (t, 1H, J=9.5 Hz, H-4), 3.50 (ddd, 1H, J₁=12.6, J₂=10.1, J₃=4.6 Hz,H-1), 3.41 (ddd, 1H, J₁=12.5, J2=9.7, J₃=4.6 Hz, H-3), 2.28 (dt, 1H,J₁=13.2, J₂=4.6 Hz, H-2eq), 1.44 (ddd, 1H, J₁=J2=J3=12.7 Hz, H-2ax);‘Ring III’: δ_(H)=5.58 (s, 1H, H-1), 5.54 (d, 1H, J=4.9 Hz, H-2), 5.44(dd, 1H, J₁=7.5, J₂=5.1 Hz, H-3), 4.31 (dd, 1H, J₁=7.1, J₂=6.0 Hz, H-4),3.67 (p, 1H, J=6.7 Hz, H-5), 1.31 (d, 3H, J=6.8 Hz, 6-CH₃); Theadditional peaks in the spectrum were identified as follow: δ_(H)=7.89(ddt, 4H, J₁=14.3, J₂=8.4, J₃=1.4 Hz, Ar), 7.57-7.50 (m, 2H, Ar),7.40-7.32 (m, 4H, Ar), 2.35 (s, 3H, CH₃), 2.10 (s, 3H, CH₃), 2.08 (s,3H, CH₃).

¹³C NMR (150 MHz, CDCl₃): δ_(C)=170.3 (CH₃—CO), 170.2 (CH₃—CO), 170.1(CH₃—CO), 165.5 (C₆H5-CO), 165.0 (C₆H5-CO), 149.3 (C5′), 133.76 (Ar),133.71 (Ar), 129.75 (Ar), 129.69 (Ar), 128.8 (Ar), 128.66 (Ar), 128.61(Ar), 128.5 (Ar), 107.2 (C1″), 97.88 (C1′), 97.87 (C4′), 80.7 (C4″),81.0 (C4), 78.2 (C5), 74.6 (C2″), 73.7 (C6), 71.9 (C3′), 66.9 (C3″),62.3 (C6′), 59.7 (C3), 59.5 (C2′), 58.8 (C5″), 58.4 (C1), 32.5 (C2),21.08 (CH₃), 21.01 (CH₃), 20.8 (CH₃), 15.6 (6″-CH₃).

Preparation of Compound 57

The glycosylation product 55 (295 mg, 0.331 mmol) was treated with asolution of MeNH₂ (33% solution in EtOH, 15 mL) and the reactionprogress was monitored by TLC (EtOAc/MeOH 85:15), which indicatedcompletion after 12 hours. The reaction mixture was evaporated todryness and was subjected to column chromatography (MeOH/EtOAc 2:8) toobtain the corresponding completely unprotected perazido derivative 57(180 mg) in 99% yield.

¹H NMR (600 MHz, MeOD): ‘Ring I’: δ_(H)=5.91 (d, 1H, J=2.6, H-1), 5.06(d, 1H, J=2.3 Hz, H-4), 4.42 (ddt, 1H, J₁=8.0, J₂=2.7, J₃=1.4, Hz, H-3),4.07-3.99 (m, 2H, H-6, H-6), 3.55 (dd, 1H, J₁=7.9, J₂=3.6 Hz, H-2);‘Ring II’: δ_(H)=3.74 (t, 1H, J=9.6 Hz, H-4), 3.66 (t, 1H, J=9.0 Hz,H-5), 3.47 (ddd, 2H, J₁=12.1, J2=8.2, J3=3.3 Hz, H-1, H-3), 3.42-3.40(m, 1H, H-6), 2.17 (dt, 1H, J₁=13.2, J₂=4.4 Hz, H-2eq), 1.28 (ddd, 1H,J₁=J2=J3=12.3 Hz, H-2ax); ‘Ring III’: δ_(H)=5.41 (d, 1H, J=1.9 Hz, H-1),4.22-4.18 (m, 2H, H-2, H-3), 3.81 (dd, 1H, J₁=9.2, J₂=3.2 Hz, H-4),3.72-3.66 (m, 1H, H-5), 1.40 (d, 3H, J=6.8 Hz, 6-CH₃);

¹³C NMR (150 MHz, MeOD): δ_(C)=152.5 (C5′), 110.5 (C1″), 100.3 (C4′),98.6 (C1′), 86.3 (C4″), 83.4 (C4), 79.4 (C4), 77.4 (C6), 76.2 (C2″),72.6 (C3″), 65.3 (C3′), 64.0 (C2′), 62.1 (C6′), 61.9 (C1), 61.7 (C3),60.6 (C5″), 33.5 (C2), 16.0 (6″-CH₃).

Preparation of NB159

To a stirred solution of Compound 57 (180 mg, 0.324 mmol) in a mixtureof THF (3 mL) and aqueous NaOH (1 mM, 5 mL), PMe₃ (1 M solution in THF,3.5 mL, 40.1 mmol) was added. The progress of the reaction was monitoredby TLC [CH₂Cl₂/MeOH/H₂O/MeNH₂ (33% solution in EtOH), 10:15:6:15], whichindicated completion after 3 hours. The reaction mixture was purified byflash chromatography on a short column of silica gel. The column waswashed with the following solvents: THF (100 mL), CH₂Cl₂ (100 mL), EtOH(50 mL), and MeOH (100 mL). The product was then eluted with the mixtureof 5% MeNH₂ solution (33% solution in EtOH) in 80% MeOH. Fractionscontaining the product were combined and evaporated under vacuum. Thepure product was obtained by passing the above product through a shortcolumn of Amberlite CG50(NH₄ ⁺ form). First, the column was washed withwater, then the product was eluted with a mixture of 10% NH₄OH in waterto yield NB159 (110 mg, 76%).

For storage and biological tests, NB159 was converted to its sulfatesalt form as follow: The free base form was dissolved in water, the pHwas adjusted to 6.7 with H₂SO₄ (0.1 N) and lyophilized to afford thesulfate salt of NB159.

¹H NMR (600 MHz, MeOD): ‘Ring I’: δ_(H)=5.39 (s, 1H, H-1), 5.01 (d, 1H,J=3.4 Hz, H-4), 4.02 (t, 1H, J=4.0 Hz, H-3), 4.0 (d, 2H, J=2.7 H-6,H-6), 3.07 (d, 1H, J=6.2 Hz, H-2); ‘Ring II’: δ_(H)=3.54 (dd, 2H,J₁=20.3, J₂=10.7 Hz, H-4, H-5), 3.18 (t, 1H, J=9.3 Hz, H-6), 2.79 (ddd,1H, J₁=12.8, J₂=6.9, J₃=4.0 Hz, H-3), 2.67 (ddd, 1H, J₁=9.6, J₂=5.1,J3=3.9 Hz, H-1), 2.04 (dt, 1H, J₁=13.1, J₂=4.3 Hz, H-2eq), 1.24 (ddd,1H, J₁=J₂=J₃=12.3 Hz, H-2ax); ‘Ring III’: δ_(H)=5.29 (s, 1H, H-1), 4.11(dd, 1H, J₁=14.7, J₂=6.2 Hz, H-2, H-3), 3.59-3.54 (m, 1H, H-4), 2.98 (t,1H, J=5.8 Hz, H-5), 1.19 (d, 3H, J=7.9 Hz, 6-CH₃);

¹³C NMR (150 MHz, MeOD): δ_(C)=153.4 (C5′), 109.8 (C1″), 100.4 (C1′),100.3 (C4′), 88.5 (C4″), 84.5 (C4), 84.0 (C5), 78.8 (C6), 76.4 (C2″),72.9 (C3″), 67.8 (C3′), 62.4 (C6′), 55.0 (C2′), 52.6 (C1), 51.4 (C3),51.3 (C5″), 37.2 (C2), 18.9 (6″-CH₃).

Readthrough Activity

Preliminary comparative in-vitro PTC suppression activity assays,performed essentially as described herein, showed that NB154 hadreadthrough activity almost 3.5 fold higher than paromamine and more orless similar activity as that of NB82.

Comparative in-vitro PTC suppression activity assays, performedessentially as described herein, further showed that NB158 and NB159both exhibit a similar or slightly lower activity compared to theircorresponding structurally related compounds NB30 and NB118.

However, the measured prokaryotic protein synthesis inhibition andsubsequently the antibacterial activity of NB154, NB158 and NB159 aresignificantly lower than that of the corresponding paromamine, NB30, andNB118, as shown in Table 6 below, suggesting that these compounds arelikely to exhibit extremely low toxicity.

TABLE 6 Antibacterial activity MIC (μg/mL) Translation Inhibition E.coli B. subtilis Compound IC₅₀ ^(Euk) (μM) IC₅₀ ^(Pro) (μM) R477/100ATCC6633 Paro. 760 ± 79 14 ± 1.2  — — NB154 375.94 ± 38.6  82.3 ±11.93 >384 >384 NB30 31 ± 4 0.45 ± 0.03  790 100 NB158 70.7 ± 2.4 36.4 ±3.5  >192 192 NB118 15.5 ± 1.3 1.9 ± 0.2  2659 83 NB159 47.9 ± 3.3 134.5± 2.8   >192 192

Example 7 Multi-Esterified Exemplary Compounds According to SomeEmbodiments of the Present Invention

An additional chemical modification on previously describedaminoglycosides was introduced with the aim of improving cellularpermeability. This modification involved the multi esterification of twoor more hydroxy groups of the aminoglycoside, to generate a pro-drugtype compound. The rational of this strategy was (i) that attaching anyhydrophobic R-group to the compound will improve its lipophylicity andas such increase the cell probability and uptake; (ii) thatintracellular esterases will hydrolyze the pro-drug to regenerate theactive drug; and (iii) that the pharmacokinetic properties of thedesired pro-drug could be improved.

Initially, three multi-esterified derivatives of G418 were synthesized:the polybenzoate derivative, Bz-G418, polyisobutyrate derivative,iBut-G418, and polyacetate derivative, Ac-G418. Bz-NB 124 was then alsosynthesized using the same synthetic protocol. Scheme 14 below Presentsthe chemical structures of these compounds.

Syntheses of Multi-Esterified G418 Compounds

Syntheses of the final compounds 63, 65 and 67 (Bz-G418, iBut-G418 andAc-G418, respectively) were performed from the commercial G418 and areillustrated in Scheme 15.

First, G418 was subjected to Boc protection on its free amine groups tothereby obtain compound 61, which serves as an intermediate for lateresterification derivatives and Boc deprotection steps via the TFA. Thechoice of the Boc protecting strategy is derived from the need toperform further selective deprotection without modifying the esterfunctional groups. The resulting final Compounds 63, 65 and 67 areconverted to TFA-addition salts, which prevent the amines to react withthe ester functionality.

Reaction of G418 and Di-tert-butyl dicarbonate yielded a mixture of per-and three-bocylated products, from which the per-bocylated product 61was isolated via column chromatography. After the isolation of compound61, the synthesis was divided to three different synthetic paths (see,Scheme 15). Esterification reactions of compound 61 with Benzoylchloride, Acetyl chloride and Isobutyryl chloride were performedseparately, to obtain the compounds 62, 64, and 66, respectively.Compound 62 was obtained upon heating the reaction mixture at 50° C. Theesterification reaction was followed by removal of the Boc protectinggroups by TFA, to thereby afford Compounds 63, 65 and 67. In all theobtained compounds the 4″ hydroxyl remained free, presumably due to alower reactivity of the tertial hydroxyl.

Preparation of Compound 61

To a stirred solution of G418 (5 grams, 10.06 mmol) in 20 mL MeOH:H₂O(1:1), Et₃N (120 mmol) was added dropwise followed by addition ofDi-tert-butyl dicarbonate (13.095 grams, 60 mmol). The reaction mixturewas heated to 50° C. and allowed to stir overnight. The propagation ofthe reaction was monitored by TLC [MeOH/EtOAc, 1:9], which indicatedcompletion after 24 hours. Thereafter, MeOH was evaporated and theremaining aqueous solution was extracted with EtOAc, washed with brineand dried over MgSO₄. Column chromatography of the residue(EtOAc/Hexane, 100% EtOAc) afforded Compound 1 as a white solid (3.96grams, 57%).

¹H NMR (500 MHz, MeOD): δ=5.45 (d, 1H, J=9.6 Hz, H-1′), 5.21 (d, 1H,J=2.3 Hz, H-1″), 4.25-4.01 (m, 4H), 3.79 (dd, J=9.9, 2.8 Hz, 1H), 3.63(t, J=8.4 Hz, 1H), 3.47 (m, 6H), 3.24-3.17 (m, 1H) 2.94 (s, 3H,NCH3-C3″), 2.14-1.92 (m, 1H,H-2), 1.44 (m, 4H,H-2, CH3-C4″), 1.24 (d,J=6.2 Hz, 3H). Additional peaks in the spectrum were identified asfollow: δ 1.44 (m, 36H, Boc).

¹³C NMR (126 MHz, MeOD): δ=159.30 (Carbamate), 159.03 (Carbamate),158.62 (Carbamate), 158.05 (Carbamate), 100.08 (C-1″), 99.09 (C-1′),82.33, 81.27 (ROC(CH₃)₃), 80.84 (ROC(CH₃)₃), 80.20 (ROC(CH₃)₃), 77.19(ROC(CH₃)₃), 75.02, 74.71, 73.70, 73.52, 73.26, 71.08, 70.93, 68.70,66.14, 61.53, 60.20, 59.00, 56.80, 28.85 (ROC(CH₃)₃), 28.84 (ROC(CH₃)₃),28.84 (ROC(CH₃)₃), 28.83 (ROC(CH₃)₃), 28.83 (ROC(CH₃)₃), 28.82(ROC(CH₃)₃), 28.79 (ROC(CH₃)₃), 28.74 (ROC(CH₃)₃), 22.58, 22.14.

MALDI TOFMS: calculated for C₄₀H₇₂N₄O₁₈ ([M+Na]⁺) m/e 919.48; measuredm/e 919.79.

Preparation of Compound 62

Compound 61 (0.6 gram, 0.668 mmol) was dissolved in anhydrous pyridine(15 mL). The solution was cooled in an ice bath under stirring andbenzoyl chloride (3 mL, 8.02 mmol) was added dropwise. The ice bath wasremoved, 4-DMAP (cat.) was added, and the reaction mixture was heated to60° C. and left overnight. The progress of the reaction was monitored byTLC (EtOAc/Hexane 5:5). After completion of the reaction as indicated byTLC, the reaction mixture was diluted with EtOAc and washed with 5% HClsolution, NaHCO₃ and brine. The combined organic layer was dried overMgSO₄, evaporated and subsequently subjected to Column chromatography ofthe residue (EtOAc/Hexane, 4:6) to thereby afford Compound 62 as a whitesolid (0.687 gram, 72%).

¹H NMR (500 MHz, CDCl₃): ‘Ring I’: δ=5.49 (d, 1H, J=4.9 Hz, H-1),4.89-4.77 (m, 2H, H-3, H-4), 4.69-4.31 (m, 1H, H-6), 4.01 (dd, 1H,J=9.8, 3.3 Hz, H-5), 3.72 (dd, 1H, J=11.4, 3.1 Hz, H-2), 1.57-0.63 (m,3H, H-7). ‘Ring II’: δ=5.45 (dd, 1H, J=7.6, 3.5 Hz, H-4), 5.19 (dd, 1H,J=14.8, 4.8 Hz, H-5), 4.08-3.96 (m, 1H, H-6), 3.24-2.98 (m, 2H,H-1,H-3), 1.92-1.66 (m, 1H, H-2 eq), 0.57-0.63 (m, 1H, H-2 ax) ‘RingIII’: δ=5.47 (dd, 1H, J=7.9, 1.5 Hz, H-2), 5.32 (d, 1H, J=4.0 Hz, H-1),4.43 (dd, 1H, J=11.6, 1.7 Hz, H-3), δ 3.44 (d, 1H, J=12.9 Hz, H-5), 2.89(s, 3H, NCH₃—C3″), 2.57 (d, 1H, J=13.2 Hz, H-5), 1.57-0.63 (m, 3H,CH₃—C4″). Additional peaks in the spectrum were identified as follow:6=8.13-7.20 (m, 25H, Ph), 1.57-0.63 (m, 36H, Boc).

¹³C NMR (126 MHz, CDCl₃): δ=165.83 (C═O), 165.75 (C═O), 165.34 (C═O),165.12 (C═O), 165.07 (C═O), 155.00 (Carbamate), 154.84 (Carbamate),154.73 (Carbamate), 154.67 (Carbamate), 133.76 (Ph), 133.66 (Ph), 133.58(Ph), 133.41 (Ph), 133.38 (Ph), 133.22 (Ph), 133.12 (Ph), 132.95 (Ph),132.90 (Ph), 130.15 (Ph), 130.03 (Ph), 129.99 (Ph), 129.96 (Ph), 129.90(Ph), 129.77 (Ph), 129.41 (Ph), 129.30 (Ph), 128.79 (Ph), 128.75 (Ph),128.67 (Ph), 128.61 (Ph), 128.54 (Ph), 128.32 (Ph), 128.23 (Ph), 128.03(Ph), 98.12 (C-1″), 96.84 (C-1′), 80.18 (C-5), 79.88 (ROC(CH₃)₃), 79.76(ROC(CH₃)₃), 79.52 (ROC(CH₃)₃), 79.44 ROC(CH₃)₃), 79.43 (ROC(CH₃)₃),79.36 (C-6′), 78.54 (C-5′) 75.84 (C-6), 73.03 (C-4), 72.39 (C-2″), 70.75(C-4′), 70.04 (C-3′), 69.05 (C-4) 69.20 (C-5″), 55.67 (s), 54.80 (C-3″),53.37 (s), 52.89 (C-3), 52.50 (C-1), 49.15 (C-2′), 49.13 (s), 49.02 (s),41.26 (NCH3-C3″), 31.54 (s), 30.32 (s), 29.62 (s), 28.44 (Boc), 28.21(Boc), 28.20 (Boc), 28.18 (ROC(CH₃)₃), 28.15 (ROC(CH₃)₃), 28.09(ROC(CH₃)₃), 28.02 (ROC(CH₃)₃), 27.89 (ROC(CH₃)₃), 27.88 (ROC(CH₃)₃),27.86 (C-6′-CH₃), 22.29, 20.84 (C-4″-CH₃).

MALDI TOFMS: calculated for C₇₅H₉₂N₄O₂₃ ([M+Na]⁺) m/e 1440.55; measuredm/e 1440.41.

Preparation of G418-Bz (63)

Compound 62 (0.687 gram, 0.523 mmol) was dissolved in freshly distilledDCM (7 mL), cooled on ice bath and TFA (2 ml) was added dropwise. Thereaction mixture was allowed to attain room temperature. Propagation ofthe reaction was monitored by TLC (Et₃N/MeOH 1:9), and indicated thecompletion of the reaction after 4 hours. The reaction mixture wasthereafter evaporated to dryness to yield G418-Bz. For storage andbiological tests, G418-Bz was dissolved in water and methanol andlyophilized to afford the TFA salt of G418-Bz (0.511 gram, 72%).

¹H NMR (500 MHz, MeOD): ‘Ring I’: δ=5.72 (dd, 1H, J=7.6, 4.8 Hz, H-3),5.60 (dd, 1H, J=6.7, 6.0 Hz, H-4), 5.47 (bs, 1H, H-1), 5.40-5.36 (m, 1H,H-6), 4.47 (dd, 1H, J=5.9, 4.6 Hz, H-5), 3.76 (dd, 1H, J=3.7, 1.6 Hz,H-2), 1.35 (d, 3H, J=3.6 Hz, H-7). ‘Ring II’: δ=5.64 (d, 1H, J=8.1 Hz,H-5), 4.55 (s, 1H, H-4), 4.30 (s, 1H, H-6), δ 3.86-3.67 (m, 2H, H-3,H-1) 2.55 (dt, 1H, J=12.5, 4.2 Hz, H-2 eq), 2.12 (q, 1H, J=12.8 Hz, H-2ax). ‘Ring III’: δ=5.34 (dd, 1H, J=10.2, 3.3 Hz, H-2), 5.29 (d, 1H,J=4.1 Hz, H-1), 3.82 (d, 1H, J=10.3 Hz, H-3), δ 3.76 (d, 1H, J=15.1 Hz,H-5), 3.13 (d, 1H, J=12.2 Hz, H-5), δ 2.89 (s, 1H), 2.89 (s, 3H,NCH3-C3″), 1.26 (s, 3H, CH₃-C4″). Additional peaks in the spectrum wereidentified as follow: δ=8.16 (d, 2H J=7.5 Hz, Ph), 8.03 (dd, 5H, J=16.4,7.5 Hz, Ph), 7.93 (d, 2H, J=7.7 Hz, Ph), 7.70 (dd, 3H, J=13.9, 7.5 Hz,Ph), 7.57 (dt, 6H, J=12.2, 5.8 Hz, Ph), 7.47-7.25 (m, 10H, Ph).

¹³C NMR (126 MHz, MeOD): δ=167.17 (C═O), 166.89 (C═O), 166.84 (C═O),166.42 (C═O), 166.41 (C═O), 163.61 (TFA), 163.33 (TFA), 163.06 (TFA),162.78 (TFA), 135.21 (Ph), 134.93 (Ph), 134.89 (Ph), 134.78 (Ph), 134.56(Ph), 134.22 (Ph), 134.03 (Ph), 131.00 (Ph), 130.98 (Ph), 130.95 (Ph),130.85 (Ph), 130.76 (Ph), 130.71 (Ph), 130.59 (Ph), 130.47 (Ph), 130.33(Ph), 130.22 (Ph), 129.95 (Ph), 129.76 (Ph), 129.67 (Ph), 129.63 (Ph),129.58 (Ph), 129.51 (Ph), 129.46 (Ph), 104.39 (C-1″), 99.84 (C-1′),83.40 (C-5), 76.05 (C-5′), 71.73 (C-2″), 71.64 (C-3′) 70.77 (C-5), 70.44(C-6), 69.05 (C-4) 68.50 (C-4′), 63.51 (C-5″), 52.46 (C-3) 50.18 (C-3″),50.10 (C-2′), 49.62 (C-1) 36.05 (NCH3-C3″), 29.15 (C-2), 22.27(C-6′-CH₃), 16.94 (C-4″-CH₃).

MALDI TOFMS: calculated for C₅₅H₆₀N₄O₁₅ ([M+H]⁺) m/e 1017.08; measuredm/e 1018.18.

Preparation of Compound 64

Compound 61 (0.5 gram, 0.557 mmol) was dissolved in anhydrous pyridine(15 mL). The solution was cooled in an ice bath under stirring andisobutyryl chloride (0.7 ml, 6.684 mmol) was added dropwise. The icebath was removed, 4-DMAP (cat.) was added, the reaction mixture washeated to 60° C. and left overnight. The Propagation of the reaction wasmonitored by TLC (EtOAc/Hexane 4:6). After the completion of thereaction as indicated by TLC, the reaction mixture was diluted withEtOAc and washed with 5% HCl solution, NaHCO₃ and brine. The combinedorganic layer was dried over MgSO₄ and evaporated. Column chromatographyof the residue (EtOAc/Hexane, 3:7) afforded Compound 64 as a white solid(0.490, 75%).

¹H NMR (500 MHz, CDCl₃): ‘Ring I’: δ=5.27 (dd, 1H, J=11.8, 3.5 Hz, H-3),5.05 (d, 1H, J=3.7 Hz, H-1), 5.00-4.96 (m, 2H, H-6, H-4), 4.59-4.54 (m,1H, H-5), 3.27 (d, 1H, J=11.9 Hz, H-2), 1.45-1.01 (m, 3H, H-7). ‘RingII’: δ=4.94 (dd, 1H, J=9.7, 8.3 Hz, H-5), 4.81-4.73 (m, 1H, H-6),4.24-4.18 (m, 1H, H-4), 4.00-3.90 (m, 1H, H-1, H-3), 1.45-1.01 (m, 2H,H-2 eq, H-2 ax). ‘Ring III’: δ=4.98 (d, 1H, J=4.7 Hz, H-1), 4.89-4.85(m, 1H, H-2), 3.55 (dd, 1H, J=11.4, 1.4 Hz, H-3), 3.38 (dd, 1H, J=4.0,2.5 Hz, H-5), 3.32 (dd, 1H, J=13.6, 1.6 Hz, H-5), 2.96 (s, 3H,NCH₃—C3″), 1.45-1.01 (m, 3H, CH₃—C4″). Additional peaks in the spectrumwere identified as follow: δ=6.76 (d, 1H, J=1.3 Hz, RNHCOOR), 5.85 (d,1H, J=2.1 Hz, RNHCOOR), 5.83 (d, 1H, J=2.3 Hz, RNHCOOR), 5.72 (d, 1H,J=4.3 Hz, RNHCOOR), 1.45-1.01 (m, 71H, Boc, i-But).

¹³C NMR (126 MHz, CDCl₃): δ=175.90 (C═O), 175.50 (C═O), 175.45 (C═O),175.39 (C═O), 174.73 (C═O), 157.49 (Carbamate), 156.54 (Carbamate),156.16 (Carbamate), 155.79 (Carbamate), 99.41 (C-1″), 99.01 (C-1′),83.09 (s), 82.29 (s), 81.73 (C-6), 79.93 (ROC(CH₃)₃), 79.79 (ROC(CH₃)₃),79.76 (ROC(CH₃)₃), 79.64 (ROC(CH₃)₃), 79.45 (C-4), 77.40 (s), 75.69 (s),74.74 (C-5), 74.42 (s), 73.50 (s), 71.20 (C-6′), 71.10 (s), 70.59(C-3″), 70.51 (s), 70.10 (C-5″), 69.73 (s), 69.05 (C-4), 68.98 (s),68.91 (C-2″), 68.37 (C-4′), 67.47 (s), 67.25 (C-3′), 65.38 (C-2′), 55.72(s), 54.52 (C-5′), 52.60 (s), 52.53 (d, J=17.3 Hz), 50.83 (C-3), 49.23(C-1), 41.20 (N—CH₃), 34.17 (i-But), 34.10 (i-But), 34.04 (i-But), 33.98(i-But), 33.74 (i-But), 29.91 (s), 29.61 (C-2), 28.40 (i-But), 28.36(i-But), 28.34 (i-But), 28.30 (i-But), 28.26 (i-But), 28.18 (i-But),22.90 (s), 18.83 (ROC(CH₃)₃), 18.73 (ROC(CH₃)₃), 18.72 (ROC(CH₃)₃),18.61 (ROC(CH₃)₃), 18.58 (ROC(CH₃)₃), 18.49 (ROC(CH₃)₃), 18.34(C-6′-CH₃), 17.91 (C-4″-CH₃).

MALDI TOFMS: calculated for C₆₀H₁₀₂N₄O₂₃ ([M+Na]⁺) m/e 1270.46; measuredm/e 1270.42.

Preparation of G418-i-But (65)

Compound 64 (0.490 gram, 0.42 mmol) was dissolved in freshly distilledDCM (10 mL), cooled on ice bath, TFA (2 mL) was added dropwise, and thereaction mixture was allowed to attain room temperature. Propagation ofthe reaction was monitored by TLC (Et3N/MeOH 1:9), which indicated thecompletion of the reaction after 4 hours. The reaction mixture wasevaporated to dryness to yield G418-i-But. For storage and biologicaltests, G418-i-But was dissolved in water and methanol and lyophilized toafford the TFA salt of G418-i-But. (0.408 gram, 78%).

¹H NMR (500 MHz, MeOD): ‘Ring I’: δ=5.38 (dd, 1H, J=8.8, 6.2 Hz, H-3),5.25 (d, 1H, J=5.9 Hz, H-1), 5.23-5.16 (m, H, H-6), 5.07 (dd, 1H, J=6.6,6.1 Hz, H-4), 4.09 (dd, 1H, J=6.0, 5.3 Hz, H-5), 3.62 (dd, 1H, J=4.6,2.0 Hz, H-2), 1.31 (d, 3H, J=6.7 Hz, H-7). ‘Ring II’: δ=5.50 (dd, 1H,J=11.3, 7.3 Hz, H-5), 4.21-4.14 (m, 2H, H-4,H-6), 3.70 (m, 2H, H-1.H-3),2.61-2.54 (m, 1H, H-2, eq), 2.17 (ddd, J=12.69, 1H, H-2,ax). ‘Ring III’:δ=5.29 (d, 1H, J=3.1 Hz, H-1), 5.21 (dd, 1H, J=8.8, 2.7 Hz, H-2), 3.66(d, 1H, J=9.7 Hz, H-3), 3.74 (d, 1H, J=13.0 Hz, H-5), 3.45 (d, 1H,J=12.5 Hz, H-5), 2.88 (s, 3H, NCH3-C3″), 1.38 (s, 3H, CH3-C4″).Additional peaks in the spectrum were identified as follow: 6=1.21 (m,35H, i-But).

¹³C NMR (126 MHz, MeOD): δ=177.35 (C═O), 177.3 (C═O 177.19 (C═O), 176.83(C═O), 176.67 (C═O), 163.22 (TFA), 162.94 (TFA), 162.66 (TFA), 162.38(TFA), 121.44 (TFA), 119.12 (TFA), 116.80 (TFA), 114.47 (TFA), 97.77(C-1″), 92.81 (C-1′), 81.81 (C-6), 77.48 (C-4), 76.61 (C-5′), 74.59(C-5), 70.03 (C-3′), 69.71 (C-6′), 69.24 (C-5″), 69.05 (C-4), 68.27(C-2″), 67.26 (C-4′), 63.62 (C-3″), 51.66 (C-2′), 50.29 (C-3), 49.86(C-1), 35.56 (NCH3-C3″), 35.22 (i-But), 35.18 (i-But), 34.93 (i-But),34.87 (i-But), 34.80 (i-But), 28.61 (C-2), 27.71 (s), 23.19 (s), 19.27(i-But), 19.24 (i-But), 19.18 (i-But), 19.16 (i-But), 19.10 (i-But),18.98 (i-But), 18.72 (i-But), 18.67 (C-6′-CH₃), 16.20 (C-4″-CH₃).

MALDI TOFMS: calculated for C₃₆H₆₃N₄O₁₃ ([M+H₂O]⁺) m/e 777.9; measuredm/e 777.54.

Preparation of Compound 66

Compound 61 (0.3 gram, 0.334 mmol) was dissolved in anhydrous pyridine(8 mL). The solution was cooled in an ice bath under stirring and acetylchloride (0.3 ml, 4.008 mmol) was added dropwise. The ice bath wasremoved, 4-DMAP (cat.) was added, and the reaction was heated to 60° C.and left overnight. The Progress of the reaction was monitored by TLC(EtOAc/Hexane 6.5:4.5). After completion of the reaction as indicated byTLC, the reaction mixture was diluted with EtOAc and washed with 5% HClsolution, NaHCO₃ and brine. The combined organic layer was dried overMgSO₄ and evaporated. Column chromatography of the residue(EtOAc/Hexane, 5:5) afforded Compound 66 as a white solid (0.25 gram,68%).

¹H NMR (500 MHz, CDCl₃): ‘Ring I’: δ=5.38 (d, 1H, J=11.5 Hz, H-1), 5.20(dd, 1H, J=2.4, 1.3 Hz, H-3), 4.60 (dd, 1H, J=11.4, 3.5 Hz, H-4), 4.45(dd, 1H, J=11.8, 1.3 Hz, H-4), 4.14-4.09 (m, 1H, H-5), 3.31 (dd, 1H,J=12.4, 1.0 Hz, H-2), 1.46-1.20 (m, 3H, H-7). ‘Ring II’: δ=3.95-3.68 (m,3H, H-4, H-5, H-6), 3.65-3.20 (m, 2H, H-1, H-3), 2.73-2.49 (m, 1H, H-2),2.46-2.26 (m, 1H, H-2). ‘Ring III’: δ=5.20-5.18 (m, 2H, H-1, H-2),4.18-4.12 (m, 1H, H-3), 3.80-3.73 (m, 1H, H-5), 3.57-3.51 (m, 1H, H-5),2.94 (s, 3H, NCH3-C3″), 1.46-1.20 (m, 3H, CH3-C4″). Additional peaks inthe spectrum were identified as follow: 6=6.74 (s, 1H, RNHCOOR), 5.90(s, 1H, RNHCOOR), 5.88 (s, 1H, RNHCOOR), 5.52 (s, 2H, RNHCOOR),2.13-1.92 (m, 12H, Ac), 1.47-1.21 (m, 36H, Boc).

¹³C NMR (126 MHz, CDCl₃): δ=170.76 (C═O), 170.59 (C═O), 169.70 (C═O),169.31 (C═O), 157.65 (Carbamate), 156.98 (Carbamate), 156.42(Carbamate), 155.63 (Carbamate), 99.48 (C-1″), 98.39 (C-1′), 85.88(C-6), 82.50 (C-4), 80.04 (s), 79.95 (ROC(CH₃)₃), 79.80 (ROC(CH₃)₃),79.64 (ROC(CH₃)₃), 79.38 (ROC(CH₃)₃), 79.29 (s), 75.66 (C-5), 74.32 (s),71.89 (C-3′), 70.58 (C-2′), 70.35 (C-5′), 70.32 (C-2″), 70.29 (C-3″),69.05 (C-4), 68.70 (C-6′), 60.35 (C-5″), 54.37 (C-4′), 50.28 (C-3),50.08 (C-1), 41.19 (N—CH₃), 30.50 (C-2), 28.38 (ROC(CH₃)₃), 28.34(ROC(CH₃)₃), 28.29 (ROC(CH₃)₃), 28.27 (ROC(CH₃)₃), 28.21 (ROC(CH₃)₃),28.14 (ROC(CH₃)₃), 22.93 (s), 21.70 (C-4″-CH₃), 20.82 (Ac), 20.72 (Ac),20.70 (Ac), 20.63 (Ac), 14.13 (C-6′-CH₃).

MALDI TOFMS: calculated for C₄₈H₈₀N₄O₂₂ ([M+Na]⁺) m/e 1088.16; measuredm/e 1088.27.

Preparation of G418-Ac (67)

Compound 66 (0.25 gram, 0.23 mmol) was dissolved in freshly distilledDCM (5 mL), cooled on ice bath and TFA (1 mL) was added dropwise. Thereaction mixture was allowed to attain room temperature. Propagation ofthe reaction was monitored by TLC (Et₃N/MeOH 1:9), which indicated thecompletion of the reaction after 4 hours. The reaction mixture wasevaporated to dryness to yield G418-Ac. For storage and biologicaltests, G418-Ac was dissolved in water and methanol and lyophilized toafford the TFA salt of G418-Ac (0.19 gram, 73%).

¹H NMR (500 MHz, MeOD): ‘Ring I’: δ=5.28 (d, 1H, J=3.8 Hz, H-1), 5.08(dd, 1H, J=10.5, 9.3 Hz, H-3), 4.67 (dd, 1H, J=10.0, 8.8 Hz, H-4),4.62-4.60 (m, 1H, H-6), 4.02 (dd, 1H, J=10.0, 1.5 Hz, H-5), 3.40 (dd,1H, J=10.9, 3.7 Hz, H-2), 0.89 (d, 3H J=6.1 Hz, H-7′). ‘Ring II’:δ=3.82-3.77 (m, 3H, H-4, H-5, H-6), 3.56-3.46 (m, 2H, H-1, H-3), 2.52(dd, 1H, J=8.0, 4.0 Hz, H-2), 2.00-1.90 (m, 1H, H-2). ‘Ring III’: δ=5.28(d, 1H, J=4.2 Hz, H-1), 5.14 (dd, 1H, J=11.2, 3.1 Hz, H-2), 3.66 (d, 1H,J=11.0 Hz, H-3), 3.90 (d, 1H, J=6.9 Hz, H-5), 3.56-3.46 (m, 1H,H-52.76), (s, 3H, NCH₃—C3″), 1.26 (s, 3H, CH₃—C4″). Additional peaks inthe spectrum were identified as follow: 6=2.08 (s, 3H, Acetate), 2.00(s, 3H, Acetate), 1.97 (s, 3H, Acetate), 1.97 (s, 3H, Acetate).

¹³C NMR (126 MHz, MeOD): δ=171.65 (carbonyl), 171.43 (carbonyl), 171.09(carbonyl), 162.93 (TFA), 162.64 (TFA), 162.36 (TFA), 162.05 (TFA),121.33 (TFA), 119.00 (TFA), 116.68 (TFA), 114.36 (TFA), 99.35 (C-1′),97.99 (C-1″), 84.02 (C-6), 83.43 (C-4), 75.15 (C-5), 73.17 (C-5′), 70.85(C-6′), 70.62 (C-2″), 70.16 (C-3′), 69.57 (C-4′), 69.09 (C-5″), 69.05(C-4), 62.74 (C-3″), 53.44 (C-2′), 49.87 (C-3), 49.73 (C-1), 35.81(N—CH₃), 29.19 (C-2), 22.05 (C-4″-CH₃), 21.04 (Acetate), 20.96(Acetate), 20.69 (Acetate), 20.50 (Acetate), 13.81 (C-7′).

MALDI TOFMS: calculated for C₂₈H₄₈N₄O₁₄ ([M+Na]⁺) m/e 664.32; measuredm/e 664.32.

Synthesis of Bz-NB124

An exemplary multi-esterified form of NB124, featuring benzyl esters andalso referred to herein as NB124-Bz ester or Bz-NB124 was prepared asdepicted in Scheme 16 below.

The starting material NB124 was synthesized by previously described[Kandasamy et al., J. Med. Chem. 2012]. NB124 as its free amine form wasfurther modified by protecting all amines by Boc-protection yieldingcompound 71. Next, the secondary hydroxyls were converted to thecorresponding benzoate esters, by treatment with benzoyl chloride,yielding compound 72. Finally, Boc-deprotection was performed bytreatment with TFA, which resulted the desired compound NB124-Bz as theTFA salt.

Preparation of Compound 71

To a stirred solution of NB124 (0.5 gram, 1.036 mmol) in 10 mL MeOH:H₂O(1:1), Et₃N (8.289 mmol) was added dropwise followed by addition ofDi-tert-butyl dicarbonate (4 grams, 18.648 mmol). The reaction washeated to 50° C. The propagation of the reaction was monitored by TLC[MeOH/EtOAc, 1:9], which indicated completion after 24 hours.Thereafter, MeOH was evaporated and the remaining aqueous solution wasextracted with EtOAc, washed with brine and dried over MgSO₄. Columnchromatography of the residue (EtOAc/Hexane, 100% EtOAc) afforded theCompound 71 as a white solid (0.580 gram, 60%).

¹H NMR (500 MHz, MeOD): δ=5.52 (s, 1H, H-1′), 5.16 (s, 1H, H-1″), 4.12(m, 3H), 3.89 (dd, J=10.0, 3.0 Hz, 1H), 3.78 (d, J=6.3 Hz, 2H),3.68-3.48 (m, 6H), 3.43 (dd, J=15.6, 7.4 Hz, 1H), 3.33-3.24 (m, 1H),1.96 (d, J=15.6 Hz, 1H), 1.51-1.47 (m, 40H, Boc) 1.27 (dd, 6H, J=10.2,4.0 Hz, C6′-CH₃, C5″-CH₃).

¹³C NMR (126 MHz, MeOD): δ=157.27 (Carbamate), 157.03 (Carbamate),156.85 (Carbamate), 156.78 (Carbamate), 109.99 (C-1″), 96.61 (C-1′),86.23, 84.35, 82.32, 79.38, 78.78, 77.17, 74.09, 73.66, 72.83, 72.30,70.49, 70.11, 69.28, 66.91, 62.86, 60.09, 55.20, 55.12, 51.03, 49.64,34.47, 29.46, 29.31, 27.46 (Carbamate), 27.44 (Carbamate), 27.33(Carbamate), 26.09, 26.07, 15.65, 13.04.

MALDI TOFMS: calculated for C₃₉H₇₀N₄O₁₈ ([M+Na]⁺) m/e 905.99; measuredm/e 905.61.

Preparation of Compound 72

Compound 71 (0.195 gram, 0.129 mmol) was dissolved in anhydrous pyridine(8 mL). The solution was cooled in an ice bath under stirring andbenzoyl chloride (0.2 ml, 1.552 mmol) was added dropwise. The ice bathwas removed, 4-DMAP (cat.) was added, and the reaction was heated to 50°C. and left overnight. The Propagation of the reaction was monitored byTLC (EtOAc/Hexane 1:1). After completion of the reaction as indicated byTLC, the reaction mixture obtained was diluted with EtOAc and washedwith 5% HCl solution, NaHCO₃ and brine. The combined organic layer wasdried over MgSO₄ and evaporated. Column chromatography of the residue(EtOAc/Hexane, 1:1) afforded Compound 70 as a white solid (0.269 gram,80%).

¹H NMR (500 MHz, CDCl₃): ‘Ring I’: δ=5.81 (d, 1H, J=4.1 Hz, H-1), 5.55(dd, 1H J=10.5, 9.0 Hz, H-3), 5.49 (dd, 1H J=5.6, 4.3 Hz, H-4),5.26-5.21 (m, 1H, H-6), 4.61 (dd, 1H, J=9.4, 2.1 Hz, H-5), 4.42 (dd, 1H,J=7.3, 1.1 Hz, H-2), 1.55 (d, 3H J=6.5 Hz, H-7). ‘Ring II’: δ=5.40 (dd,1H, J=3.6, 2.0 Hz, H-4), 5.27 (dd, 1H, J=3.5, 1.4 Hz, H-5), 4.13-4.05(m, 1H, H-6), 3.93-3.84 (m, 2H, H-1,H-3), 1.55 (dd, 1H, J=4.9, 1.1 Hz,H-2 eq), 1.24-1.20 (m, 1H, H-2 ax). ‘Ring III’: δ=5.39 (d, 1H, J=4.7 Hz,H-1), 5.28 (dd, 1H, J=12.8, 6.1 Hz, H-3), 5.10 (dd, 1H, J=9.5, 4.6 Hz,H-2), 3.76-3.64 (m, 1H, H-4), 1.55 (d, 1H, J=6.5 Hz, H-5), 1.22 (d, 3H,J=6.3 Hz, H-6). Additional peaks in the spectrum were identified asfollow: 6=8.14-7.13 (m, 30H, Ph), 1.51 (s, 9H, Boc), 1.39 (s, 9H, Boc),1.25 (s, 9H, Boc), 1.12 (s, 9H, Boc).

¹³C NMR (101 MHz, CDCl₃): δ=166.39 (C═O), 165.94 (C═O), 165.49 (C═O),165.36 (C═O), 164.94 (C═O), 164.41 (C═O), 155.59 (Carbamate), 155.28(Carbamate), 155.05 (Carbamate), 154.78 (Carbamate), 133.44 (Ph), 133.36(Ph), 133.23 (Ph), 133.05 (Ph), 132.95 (Ph), 130.38 (Ph), 130.24 (Ph),129.96 (Ph), 129.88 (Ph), 129.82 (Ph), 129.55 (Ph), 129.31 (Ph), 129.11(Ph), 128.96 (Ph), 128.87 (Ph), 128.78 (Ph), 128.29 (Ph), 128.22 (Ph),128.13 (Ph), 107.44 (C-1″), 97.30 (C-1′), 82.93 (C-6), 81.67, 80.03(ROC(CH₃)₃), 79.56 (ROC(CH₃)₃), 79.40 (ROC(CH₃)₃), 79.19 (ROC(CH₃)₃),78.32 (C-3), 75.77 (C-4), 75.68 (C-3″) 75.15 (C-5), 72.48 (C-4′), 70.02(C-2″), 70.69 (C-6′), 70.07 (C-5′), 69.94 (C-3′), 60.33 (C-4″), 53.26(C-2′), 50.2 (C-5″) 49.68 (C-3), 47.64 (C-1), 34.82 (C-2), 28.47(ROC(CH₃)₃), 28.42 (ROC(CH₃)₃), 27.95 (ROC(CH₃)₃), 27.80 (ROC(CH₃)₃),20.97 (C-7′), 17.73 (C-6″), 14.18 (C-6″).

MALDI TOFMS: calculated for C₈₁H₉₄N₄O₂₄ ([M+Na]⁺) m/e 1530.81; measuredm/e 1530.81.

Preparation of NB124-Bz

Compound 72 (0.189 gram, 0.125 mmol) was dissolved in freshly distilledDCM (5 mL), cooled on ice bath and TFA (1 mL) was added dropwise. Thereaction mixture was allowed to attain room temperature. Propagation ofthe reaction was monitored by TLC (Et3N/MeOH 1:99), which indicated thecompletion of the reaction after 4 hours. The reaction mixture wasevaporated to dryness to yield NB124-Bz. For storage and biologicaltests, NB124-Bz was dissolved in water and methanol and lyophilized toafford the TFA salt of NB124-Bz (0.191 gram, 97%).

¹H NMR (500 MHz, MeOD): ‘Ring I’: δ=6.53 (d, 1H, J=3.8 Hz, H-1), 6.10(dd, 1H J=10.6, 9.9 Hz, H-3), 5.76 (dd, 1H J=10.1, 9.2 Hz, H-4),5.53-5.47 (m, 1H, H-6), 4.53 (dd, 1H, J=9.8, 3.9 Hz, H-5), 4.09 (d, 1HJ=13.5 Hz, H-2), 1.46 (d, 3H, J=3.2 Hz, H-7). ‘Ring II’: δ=5.51 (dd, 1H,J=10.0, 8.6 Hz, H-4), 4.85-4.81 (m, 1H, H-5), 4.47 (dd, 1H, J=10.1, 8.2Hz, H-6), 3.88-3.77 (m, 2H, H-1, H-3), 2.68-2.61 (m, 1H, H-2), 2.19-2.09(m, 1H, H-2). ‘Ring III’: δ=5.77 (s, 1H, H-1), 5.50 (dd, 1H, J=8.8, 4.3Hz, H-3), 5.40 (dd, 1H, J=3.5, 0.4 Hz, H-2), 4.10 (dd, 1H, J=10.0, 8.6Hz, H-4), 3.52-3.46 (m, 1H, H-5), 0.96 (d, 3H, J=6.6 Hz, H-6). Theadditional peaks in the spectrum were identified as follow: 6=8.03-7.81(m, Ph), 7.65-7.24 (m, Ph), 7.01 (m, Ph).

¹³C NMR (126 MHz, MeOD): δ=167.31 (C═O), 167.13 (C═O), 166.92 (C═O),166.76 (C═O), 165.67 (C═O), 165.14 (C═O), 163.27 (TFA), 162.99 (TFA),162.70 (TFA), 162.43 (TFA), 135.04 (Ph), 134.91 (Ph), 134.90 (Ph),134.87 (Ph), 134.84 (Ph), 134.57 (Ph), 134.03 (Ph), 130.97 (Ph), 130.82(Ph), 130.70 (Ph), 130.51 (Ph), 129.95 (Ph), 129.86 (Ph), 129.69 (Ph),129.67 (Ph), 129.60 (Ph), 129.58 (Ph), 129.50 (Ph), 129.45 (Ph), 129.34(Ph), 129.24 (Ph), 129.02 (Ph), 121.29 (TFA), 118.98 (TFA), 116.66(TFA), 114.35 (TFA), 107.23 (C-1″), 92.70 (C-1′), 81.08 (C-5), 80.78(C-2′), 76.93 (C-6), 76.76 (C-2″), 75.28 (C-4), 72.95 (C-3″), 72.48(C-5′), 71.82 (C-3′), 71.48 (C-6′), 70.92 (C-4′), 53.85 (C-4″), 52.34(C-5″), 50.26 (C-3), 50.04 (C-1), 29.28 (C-2), 16.61 (C-7′), 14.19(C-6″).

MALDI TOFMS: calculated for C₆₁H₆₂N₄O₁₆ ([M+Na]⁺) m/e 1129.42; measuredm/e 1129.42.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention. To the extent thatsection headings are used, they should not be construed as necessarilylimiting.

What is claimed is:
 1. A compound having the following chemical formula:


2. A pharmaceutical composition comprising the compound of claim 1 and apharmaceutically acceptable carrier.
 3. A method of treating a geneticdisorder in a subject in need thereof, the method comprisingadministering to the subject a therapeutically effective amount of thecompound of claim
 1. 4. The method of claim 3, wherein said geneticdisorder is associated with a premature stop-codon truncation mutation.5. The method of claim 3, wherein said genetic disorder is selected fromthe group consisting of cystic fibrosis (CF), Duchenne musculardystrophy (DMD), ataxia-telangiectasia, Hurler syndrome, hemophilia A,hemophilia B, Usher syndrome, Tay-Sachs, Becker muscular dystrophy(BMD), Congenital muscular dystrophy (CMD), Factor VII deficiency,Familial atrial fibrillation, HaileyHailey disease, McArdle disease,Mucopolysaccharidosis, Nephropathic cystinosis, Polycystic kidneydisease, Rett syndrome, Spinal muscular atrophy (SMA), cystinosis,Severe epidermolysis bullosa, Dravet syndrome, X-linked nephrogenicdiabetes insipidus (XNDI), X-linked retinitis pigmentosa and cancer.